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Results for “germ layer specification”

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At least 163 records · Page 9Linked to original sources
PubMed2006

Effect of leukemia inhibitory factor on embryonic stem cell differentiation: implications for supporting neuronal differentiation.

AIM: Leukemia inhibitory factor (LIF), a pleiotropic cytokine, has been used extensively in the maintenance of mouse embryonic stem cell pluripotency. In this current work, we examined the effect of the LIF signaling pathway in embryonic stem (ES) cell differentiation to a neural fate. METHODS: In the presence of LIF (1000 U/mL), the production of neuronal cells derived from embryoid bodies (EB) was tested under various culture conditions. Inhibition of the LIF pathway was examined with specific inhibitors. The effects of cell apoptosis and proliferation on neural differentiation were examined. ES cell differentiation into three-germ layers was compared. RESULTS: Under various culture conditions, neuronal differentiation was increased in the presence of LIF. Blocking the LIF-activated STAT3 signaling pathway with specific inhibitors abolished the neuronal differentiation of ES cells, whereas inhibition of the LIF-activated MEK signaling pathway impaired the differentiation of ES cells toward a glial fate. LIF suppressed cell apoptosis and promoted cell proliferation during ES cell differentiation. LIF inhibited the differentiation of ES cells to both mesoderm and extraembryonic endoderm fates, but enhanced the determination of neural progenitors. CONCLUSION: These results suggest that LIF plays a positive role during the differentiation of ES cells into neuronal cells.

Animals↗
PubMed2000

goosecoid expression represses Brachyury in embryonic stem cells and affects craniofacial development in chimeric mice.

The homeobox gene goosecoid, originally identified in Xenopus, is expressed in the organizer or its equivalent during gastrulation in the frog, chick, zebrafish and mouse. To investigate the role of goosecoid in mouse development, we have generated embryonic stem cells that stably overexpress the murine homolog of goosecoid. These cells show a repression of the gastrulation-associated gene Brachyury. Interestingly, repression of Brachyury is conserved between Xenopus and mouse despite the lack of conservation of the Brachyury promoter. Further characterization of the goosecoid-overexpressing ES cells revealed that they maintain the expression of stage-specific embryonic antigen-1, and teratomas derived from goosecoid-overexpressing cells show the presence of cell types derived from all three germ layers. Some highly chimeric mice derived from goosecoid-overexpressing cells displayed skull defects. These observations suggest that goosecoid may play a role in specification of anterior mesendodermal fates and specifically in mouse craniofacial development.

Animals↗
PubMed1987

Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development.

This paper shows stage- and tissue-specific global demethylation and remethylation occurring during embryonic development. The egg genome is strikingly undermethylated and the sperm genome relatively methylated. Following a loss of genomic methylation during preimplantation development, embryonic and extraembryonic lineages are progressively and independently methylated to different final extents. Methylation continues postgastrulation and hence could be a mechanism initiating, or confirming, differential programming in the definitive germ layers. It is proposed that much of the methylation observed in somatic tissues acts to stabilize and reinforce prior events that regulate the activity of specific genes, chromosome domains or the X chromosome (in females). Fetal germ cell DNA is markedly undermethylated and we favour the idea that the germ lineage is set aside before the occurrence of extensive methylation of DNA in fetal precursor cells.

Animals↗
PubMed1997

Mesoderm is required for the formation of a segmented endodermal cell layer in the leech Helobdella.

The homeobox gene Lox3 is expressed in a segmentally iterated pattern within the endoderm of the leech Helobdella. We use that expression here to study endoderm differentiation following experimental ablations of mesoderm. Lox3 RNA was first detected by in situ hybridization at the stage when a definitive cellular endoderm is formed from its syncytial precursor and was never observed in derivatives of other germ layers. Expression is initially distributed throughout the endoderm, but rapidly disappears from specific regions of the nascent gut wall so as to produce a pattern of segmental stripes. The stripe pattern differs markedly between midgut organs, with thin stripes of Lox3 expression in the intercaecal constrictions of the crop and wide stripes of Lox3 expression marking the caecal bulges of the intestine. Lox3 expression in the rectum is not obviously segmental. Ablation of segmental mesoderm in the early Helobdella embryo prevents the formation of definitive endoderm and the expression of Lox3 RNA and leads to abnormalities in the morphogenesis of the gut tube. These endodermal deficits are precisely coextensive with the zone of mesodermal deficiency, suggesting that the mesoderm normally acts to promote the formation of the endodermal cell layer via local cell interactions. The segmental pattern of Lox3 expression is largely unaffected in portions of the endoderm surrounding such deficits, suggesting that endodermal segmentation is not established by lateral interactions within that tissue layer. Rather, we propose that the segmental organization of the endoderm is imprinted by vertical interactions with the segmental mesoderm.

Amino Acid Sequence↗
PubMed1999

EHD1--an EH-domain-containing protein with a specific expression pattern.

A cDNA that is a member of the eps15 homology (EH)-domain-containing family and is expressed differentially in testis was isolated from mouse and human. The corresponding genes map to the centromeric region of mouse chromosome 19 and to the region of conserved synteny on human chromosome 11q13. Northern analysis revealed two RNA species in mouse. In addition to the high levels in testis, expression was noted in kidney, heart, intestine, and brain. In human, three RNA species were evident. The smaller one was predominant in testis, while the largest species was evident in other tissues as well. The predicted protein sequence has an EH domain at its C-terminus, including an EF, a Ca2+ binding motif, and a central coiled-coil structure, as well as a nucleotide binding consensus site at its N-terminus. As such, it is a member of the EH-domain-containing protein family and was designated EHD1 (EH domain-containing 1). In cells in tissue culture, we localized EHD1 as a green fluorescent protein fusion protein, in transferrin-containing, endocytic vesicles. Immunostaining of different adult mouse organs revealed major expression of EHD1 in germ cells in meiosis, in the testes, in adipocytes, and in specific retinal layers. Results of in situ hybridization to whole embryos and immunohistochemical analyses indicated that EHD1 expression was already noted at day 9.5 in the limb buds and pharyngeal arches and at day 10.5 in sclerotomes, at various elements of the branchial apparatus (mandible and hyoid), and in the occipital region. At day 15.5 EHD1 expression peaked in cartilage, preceding hypertrophy and ossification, and at day 17.5 there was no expression in the bones. The EHD1 gene is highly conserved between nematode, Drosophila, mouse, and human. Its predicted protein structure and cellular localization point to the possibility that EHD1 participates in ligand-induced endocytosis.

Amino Acid Sequence↗
PubMed1999

Confocal microscopic analysis of morphogenetic movements.

Confocal microscopy is an excellent means of imaging cellular dynamics within living zebrafish embryos because it provides a means of optically sectioning tissues that have been labeled with specific fluorescent probe molecules. In order to study genetically encoded patterns of cell behavior that are involved in the formation of germ layers and various organ primordia, it is possible to vitally stain an entire zebrafish embryo with one or more fluorescent probe molecules and then examine morphogenetic behaviors within specific cell populations of interest using time-lapse confocal microscopy. There are two major advantages to this "bulk-labeling" approach: (1) the applied fluorescent probe (a contrast-enhancing agent) allows all of the cells within an intact zebrafish embryo to be rapidly stained; (2) the morphogenetic movements and shape changes of hundreds of cells can then be examined simultaneously in vivo using time-lapse confocal microscopy. The neutral fluorophore Bodipy 505/515 and its sphingolipid-derivative Bodipy-C5-ceramide are particularly useful, nonteratogenic vital stains for imaging cellular dynamics in living zebrafish embryos. These photostable fluorescent probes (when applied with 2% DMSO) percolate through the enveloping layer epithelium of the embryo, and localize in yolk-containing cytoplasm and interstitial space, respectively, owing to their different physiochemical characteristics. Bodipy-ceramide, for instance, remains highly localized to interstitial fluid once it accumulates within a zebrafish embryo, allowing the boundaries of deep cells to be clearly discerned throughout the entire embryo. Through the use of either of these fluorescent vital stains, it is possible to rapidly convert a developing zebrafish embryo into a strongly fluorescent specimen that is ideally suited for time-lapse confocal imaging. For zebrafish embryos whose deep cells have been intentionally "scatter-labeled" with fluorescent lineage tracers (e.g., fluorescent dextrans), sequential confocal z-series (i.e., focus-throughs) of the embryo can be rendered into uniquely informative 3D time-lapse movies using readily available image-processing programs. Similar time-lapse imaging, combined with rapidly advancing computer-assisted visualization techniques, may soon be applied to study the dynamics of GFP-fusion proteins in vivo, as well as other types of synthetic probe molecules designed to reveal the cytological processes associated with the patterning and morphological transformations of the zebrafish's embryonic tissues.

Animals↗
PubMed2000

The EGF-CFC gene family in vertebrate development.

EGF-CFC genes encode extracellular proteins that play key roles in intercellular signaling pathways during vertebrate embryogenesis. Mutations in zebrafish and mouse EGF-CFC genes lead to defects in germ-layer formation, anterior-posterior axis orientation and left-right axis specification. In addition, members of the EGF-CFC family have been implicated in carcinogenesis. Although formerly regarded as signaling molecules that are distant relatives of epidermal growth factor (EGF), recent findings indicate that EGF-CFC proteins act as essential cofactors for Nodal, a member of the transforming growth factor beta (TGF-beta) family. Here, we review molecular genetic evidence from mouse and zebrafish on biological and biochemical roles of the EGF-CFC family, and discuss differing models for EGF-CFC protein function.

Amino Acid Sequence↗
PubMed2005

Winged-helix transcription factors and pancreatic development.

The forkhead gene family, named after the founding gene member in Drosophila, is characterized by a unique DNA-binding domain. This so-called forkhead box encodes a winged-helix DNA-binding motif, the name of which describes the structure of the domain when bound to DNA. The three Fox (forkhead box) group A genes, Foxa1, Foxa2 and Foxa3, are expressed in embryonic endoderm, the germ layer that gives rise to the digestive system, and contribute to the specification of the pancreas and the regulation of glucose homoeostasis. Deletion of the Foxa2 gene in pancreatic beta-cells in mice results in a phenotype resembling PHHI (persistent hyperinsulinaemic hypoglycaemia of infancy). Molecular analyses have demonstrated that Foxa2 is an important regulator of the genes encoding Sur1, Kir6.2 and Schad (short chain L-3-hydroxyacyl-CoA dehydrogenase), mutation of which causes PHHI in humans. Foxa1 was shown to be an essential activator of glucagon gene expression in vivo. An additional winged-helix protein, Foxo1, contributes to pancreatic beta-cell function by regulating the Pdx1 gene, which is required for pancreatic development in cooperation with Foxa2.

Animals↗
PubMed2005

Comparison of neurosphere cells with cumulus cells after fusion with embryonic stem cells: reprogramming potential.

Embryonic stem cells (ESCs) are the pluripotent cells that also have the capacity to induce the genomic reprogramming of differentiated somatic cells. The progressively restricted genomic potential of somatic cells observed during embryonic development can be reverted to a pluripotent state by the formation of cell hybrids with ESCs. To assess the reprogramming potential of ESCs, we investigated the reprogramming of one of two different somatic cell populations, neurosphere cells (NSCs) and cumulus cells (CCs), after fusion with ESCs. Specifically, hybrid cells were produced by cell fusion of E14 ESCs with either NSCs or CCs containing the neo/lacZ and Oct4-GFP transgenes. The first reprogramming event, observed by the presence of Oct4-GFP in the hybrid cells, could be identified on Day 2, at approximately 45 h after fusion in both ESC-NSC and ESC-CC hybrids. In addition, the two ESC-somatic cell hybrids exhibit a similar reprogramming rate and share characteristics with the E14 ESC line: (1) expression of pluripotent markers (Oct4, Rex-1 and nanog); (2) inactivation of differentiated tissue-specific gene expression; and (3) the capacity to differentiate into all three germ layers. Taken together, our results suggest that the ESC-somatic cell hybrids have fully acquired ESC characteristics and that somatic cells of different tissue origin have the same potential to be reprogrammed after fusion with ESCs.

Animals↗
PubMed2005

Human embryonic stem cells: possibilities for human cell transplantation.

Human embryonic stem (ES) cells serve as a potentially unlimited renewable source for cell transplantation targeted to treat several diseases. One advantage of embryonic stem (ES) cells over other stem cells under research is their apparently indefinite self-renewal capacity if cultured appropriately, and their ready differentiation into various cell phenotypes of all three germ layers. To date, a number of studies have reported the derivation of specific functional derivatives from human ES cells in vitro. While there have been clinical trials of human embryonal carcinoma (EC) cell-derived neurons in humans there has been no attempt as yet using human ES cell derivatives. However, the latter have been transplanted into recipient animals. In some cases ES-derived cells were shown to undergo further maturation, displayed integration with host tissue and even ameliorated the disease condition in the animal model. Recently, it has been reported that human ES cells can be genetically manipulated. Such procedures could be used to direct differentiation to a specific cell type or to reduce graft rejections by the modification of immune responses. This review highlights some of the recent advances in the field and the challenges that lie ahead before clinical trials using ES-derived cells can be contemplated.

Cell Culture Techniques↗
PubMed2002

Neural stem cells: plasticity and their transdifferentiation potential.

The presence of resident stem cells in adult tissues is of fundamental importance for the maintenance of their structural and functional integrity. In fact, throughout life, somatic stem cells attend to the critical function of substituting terminally differentiated cells lost to physiological turnover, injury or disease. Thence, one of the basic dogmata in tissue biology holds that the differentiation potential of an adult stem cell is restricted to the generation of the mature cell lineages found in the tissue to which the stem cell belongs. A plethora of recent evidences from many groups, including ours, is now providing evidence that adult stem cells may possess a broader differentiation repertoire than expected and that their fate potential may not be as tissue specific as once thought. The initial example of an unforeseen, trans-germ layer plasticity - that seems now to emerge as a prototypic functional trait of various somatic stem cells of different origin - has come from the reported awakening of a latent hemopoietic developmental capacity in stem cells isolated from the adult mammalian brain following their transplantation into sub-lethally irradiated mice. More recently, it has been shown that adult neural stem cells can differentiate into a wide array of bodily cells of different origin when injected into the blastocyst and into myogenic cells when transplanted into the adult regenerating skeletal muscle. Moreover, bone marrow stem cells can now give rise to skeletal muscle, hepatic and brain cells, whereas muscle precursors can generate blood cells. In this article, we review some of the basic notions regarding the functional properties of the adult neural stem cells and discuss findings in the expanding area of trans-germ layer conversion, with emphasis on the neural stem cell.

Animals↗
PubMed1982

Reactivity of human lymphocytes to teratoma preparations as revealed by the macrophage electrophoretic mobility (MEM) technique.

By means of the MEM technique, the lymphokine release by human lymphocytes has been measured after exposure to 3M KCl extracts from malignant teratoma tissue. Clear-cut responses were found in 149 out of 171 cancer patients (87%) regardless of the primary cancer site. In contrast, only 15 out of 93 (16%) individuals from a control group without detectable malignant disease gave such a response. Subdivision of that control group showed, however, that only 1 of 46 healthy blood donors, but 14 of 47 (30%) hospital patients with benign tumours (7 of 19), inflammatory diseases or degenerative and metabolic disorders (7 of 28) reacted to the teratoma-derived material. It appears to be possible that malignant teratoma tissues contain a mixture of oncofetal or phase-specific antigens which correspond to the multifarious differentiation structures of the three germ layers developing in such tumours. If the lymphocyte response of tumour patients is due to the sensitization to analogous tumour-associated antigens in vivo, it remains to be clarified whether or not the less frequent lymphocyte response of hospital patients without neoplastic diseases may be directed to another quality of antigens.

Adolescent↗
PubMed2003

Early endoderm development in vertebrates: lineage differentiation and morphogenetic function.

Gastrulation of the vertebrate embryo culminates in the formation of three primary germ layers: ectoderm, mesoderm and endoderm. The endoderm contributes to the lining of the gut and the associated organs. New components of the molecular pathway for endoderm specification have been identified in the zebrafish and Xenopus. In the mouse, the activity of orthologous factors is involved with the allocation and differentiation of the definitive endoderm. Morphogenetic interactions between the endoderm and the other germ layer derivatives are critical for the morphogenesis of head structures and organogenesis of gut derivatives.

Animals↗
PubMedDate not supplied

Immunostaining of stage-specific embryonic antigen-4 in intratubular germ cell neoplasia unclassified and in testicular germ-cell tumors.

Stage-specific embryonic antigen-4 (SSEA-4) is expressed in testicular germ-cell tumors (GCT) according to studies using thin-layer chromatography (TLC) immunostaining. To further understand the relationship between SSEA-4 and the histogenesis of testicular GCT, we examined the expression of SSEA-4 in 43 samples of testicular GCT either by immunohistochemical staining or by TLC immunostaining. Immunohistochemical staining detected SSEA-4 in spermatogonia from the non-tumor parts of the testis and in all of 8 samples of intratubular germ cell neoplasia unclassified (IGCNU). Immunohistochemical staining was SSEA-4 positive in all 9 seminomas, and in one yolk sac tumor and in 5 embryonal carcinomas among 9 non-seminomas. Immunostaining with TLC showed that SSEA-4 was retained in 15 of 16 seminomas and in 4 of 8 non-seminomas. These results demonstrate an antigenic link between spermatogonia, IGCNU, and testicular GCT. We suggest that SSEA-4 is associated with the histogenesis of testicular GCT.

Adult↗
PubMed2005

Embryonic even skipped-dependent muscle and heart cell fates are required for normal adult activity, heart function, and lifespan.

The Drosophila pair-rule gene even skipped (eve) is required for embryonic segmentation and later in specific cell lineages in both the nervous system and the mesoderm. We previously generated eve mesoderm-specific mutants by combining an eve null mutant with a rescuing transgene that includes the entire locus, but with the mesodermal enhancer removed. This allowed us to analyze in detail the defects that result from a precisely targeted elimination of mesodermal eve expression in the context of an otherwise normal embryo. Absence of mesodermal eve causes a highly selective loss of the entire eve-expressing lineage in this germ layer, including those progeny that do not continue to express eve, suggesting that mesodermal eve precursor specification is not implemented. Despite the resulting absence of a subset of muscles and pericardial cells, mesoderm-specific eve mutants survive to fertile adulthood, providing an opportunity to examine the effects of these developmental abnormalities on adult fitness and heart function. We find that in these mutants, flying ability, myocardial performance under normal and stressed conditions, and lifespan are severely reduced. These data imply a nonautonomous role of the affected pericardial cells and body wall muscles in developing and/or maintaining cardiac performance and possibly other functions contributing to normal lifespan. Given the similarities of molecular-genetic control between Drosophila and vertebrates, these findings suggest that peri/epicardial influences may well be important for proper myocardial function.

Aging↗
PubMed2004

Bioreactor cultivation enhances the efficiency of human embryoid body (hEB) formation and differentiation.

The promise of human embryonic stem cells (hESCs) to provide an unlimited supply of cells for cell therapy and tissue engineering depends on the availability of a controllable bioprocess for their expansion and differentiation. We describe for the first time the formation of differentiating human embryoid bodies (hEBs) in rotating bioreactors to try and control their agglomeration. The efficacy of the dynamic process compared to static cultivation in Petri dishes was analyzed with respect to the yield of hEB formation and differentiation. Quantitative analyses of hEBs, DNA and protein contents, and viable cell concentration, as measures for culture cellularity and scale-up, revealed 3-fold enhancement in generation of hEBs compared to the static culture. Other metabolic indices such as glucose consumption, lactic acid production, and pH pointed to efficient cell expansion and differentiation in the dynamic cultures. The type of rotating vessel had a significant impact on the process of hEB formation and agglomeration. In the slow turning lateral vessel (STLV), hEBs were smaller in size and no large necrotic centers were seen, even after 1-month cultivation. In the high aspect rotating vessel (HARV), hEB agglomeration was massive. The appearance of representative tissues derived from the three germ layers as well as primitive neuronal tube organization, blood vessel formation, and specific-endocrine secretion indicated that the initial developmental events are not altered in the dynamically formed hEBs. Collectively, our study defines the culture conditions in which control over the aggregation of differentiating hESCs is obtained, thus enabling scaleable cell production for clinical and industrial applications.

Apoptosis↗
PubMed1982

Development of a latex-conjugated immunocytological marker for scanning electron microscopic analysis of quail-chick chimeras.

Quail-chick chimeras are created by transplanting pieces of quail tissue to chick. We have used antibodies combined with a cell surface marking technique [modified after Molday et al.('75)], to facilitate scanning electron microscopic (SEM) identification of quail cells in such chimeras. First, antibodies to quail RBCs were raised in rabbits by intravenous injection. Rabbit anti-quail RBC serum was precipitated by ammonium sulfate, purified by DEAE chromatography, and cross-absorbed with chicken RBCs. Second, sheep anti-rabbit IgG was purchased commercially and further purified by affinity chromatography. Third, 900-A-diameter latex beads were synthesized by aqueous emulsion copolymerization of methacrylates. Spheres were bound with diaminoheptane to create an extension arm which was further derivatized in a two-step glutaraldehyde procedure. Purified sheep IgG was bound to the aldehyde-activated spheres, with uncoupled sheep IgG removed by sucrose density centrifugation. To test the marker, rabbit anti-quail IgG was added to 1% diluted quail RBCs. After washing, sheep anti-rabbit IgG bound spheres were introduced. Washed cells were fixed in one-half strength Karnovsky's and processed for SEM. Quail RBCs were uniformly decorated with beads, containing 2,000 beads per cell. Similarly treated chick RBCs show no binding to beads. Likewise, quail RBCs not pre-treated with the rabbit IgG do not bind beads. Prefixed quail RBCs still bind latex-conjugated beads, although at somewhat reduced levels. When mixtures of quail and chick RBCs were processed for identification: (1) sphere labeling was an "all or none" phenomenon; (2) the proportion of bead-decorated cells observable in the SEM was the same as the proportion of quail RBCs provided in the initial mix; and (3) morphologically distinguishable embryonic chick RBCs did not label whereas under the same conditions, quail RBCs do. We further demonstrate that rabbit antibodies prepared by injection of stage 4 quail primitive streaks can be used to specifically label quail epiblast and mesoblast cells, providing markers for at least two germ layers. It is now possible to combine grafting techniques of known success, with SEM analysis of the chimera.

Animals↗