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Depletion of the maternal store of the localised mRNA encoding the T-box transcription factor VegT in Xenopus embryos has recently been shown to dramatically block endoderm formation and change the normal position of the mesodermal and ectodermal germ layers.
BACKGROUND: In Xenopus, the endoderm germ layer is derived from the vegetal blastomeres of cleavage-stage embryos. Cell transplantation experiments have revealed that the endodermal fate becomes gradually fixed during the late blastula stages. Sox17alpha, Mix.1, Mixer and GATA-4 encode vegetal zygotic transcription factors with endoderm-inducing activity. The accumulation of their transcripts during the late blastula stages may cause determination of the endodermal fate. VegT, a T-box transcription factor, the maternal transcripts of which are vegetally localised, is also required for endoderm formation. RESULTS: We analysed the events leading to the progressive accumulation of the transcripts for Sox17alpha, Mix.1, Mixer and GATA-4. Two phases could be distinguished in the endodermal programme. In phase 1, Sox17alpha, Mix.1, and the genes encoding transforming growth factor beta-related signalling molecules Xnr1, Xnr2 and Derrière were activated cell-autonomously at around the mid-blastula transition (MBT) by maternal determinants. In phase 2, TGFbeta signalling, possibly involving Xnr1, Xnr2 and Derrière, led to the activation of Mixer and GATA-4 in late blastula stages and to the reinforcement of the expression of Sox17alpha and Mix.1. Overexpression of VegT in animal caps triggered a developmental programme qualitatively similar to that observed in vegetal blastomeres, except that Xnr1 and GATA-4 were not activated by the early gastrula stage. CONCLUSIONS: Our results support a two-step model for endoderm determination between fertilisation and the onset of gastrulation. The initial cell-autonomous activation of early endodermal genes by maternal determinants including, but not limited to, VegT is relayed by the action of zygotic TGFbetas such as Xnr1, Xnr2 and Derrière.
BACKGROUND: The establishment, maintenance and rearrangement of junctions between epithelial cells are extremely important in many developmental, physiological and pathological processes. AF-6 is a putative Ras effector; it is also a component of tight and adherens junctions, and has been shown to bind both Ras and the tight-junction protein ZO-1. In the mouse, AF-6 is encoded by the Af6 gene. As cell-cell junctions are important in morphogenesis, we generated a null mutation in the murine Af6 locus to test the hypothesis that lack of AF-6 function would cause epithelial abnormalities. RESULTS: Although cell-cell junctions are thought to be important in early embryogenesis, homozygous mutant embryos were morphologically indistinguishable from wild-type embryos through 6.5 days post coitum (dpc) and were able to establish all three germ layers. The earliest morphological abnormalities were observed in the embryonic ectoderm of mutant embryos at 7.5 dpc. The length of the most apical cell-cell junctions was reduced, and basolateral surfaces of those cells were separated by multiple gaps. Cells of the embryonic ectoderm were less polarized as assessed by histological criteria and lateral localization of an apical marker. Mutant embryos died by 10 dpc, probably as a result of placental failure. CONCLUSIONS: AF-6 is a critical regulator of cell-cell junctions during mouse development. The loss of neuroepithelial polarity in mutants is consistent with a loss of efficacy of the cell-cell junctions that have a critical role in establishing apical/basolateral asymmetry.
The conversion of an epithelial monolayer into a multilayered structure consisting of the three germ layers, ectoderm, mesoderm and endoderm, constitutes a conserved theme in the early development of animals. This is accomplished by morphogenetic movements that occur during gastrulation and serve not only to generate shape but also to ensure that cells receive the right signals at the right time. Recent evidence of the role of molecular interactions facilitated by cell movements in continuously defining the chick 'organizer' during gastrulation challenges the notion that it is a fixed cell population derived from an exclusive cell lineage.
Pax genes, which encode a family of transcription factors, are essentially required for the formation of several tissues from all germ layers in the mammalian embryo. Specifically, in organogenesis, they are involved in triggering early events of cell differentiation. The differentiation of endoderm-derived endocrine pancreas is mediated through Pax4 and Pax6. In the thyroid gland, Pax8 is essential for the formation of thyroxine-producing follicular cells, also of endodermal origin. The analysis of loss-of-function mutants revealed a common function of Pax genes in organogenesis.
During vertebrate gastrulation, large cellular rearrangements lead to the formation of the three germ layers, ectoderm, mesoderm and endoderm. Zebrafish offer many genetic and experimental advantages for studying vertebrate gastrulation movements. For instance, several mutants, including silberblick, knypek and trilobite, exhibit defects in morphogenesis during gastrulation. The identification of the genes mutated in these lines together with the analysis of the mutant phenotypes has provided new insights into the molecular and cellular mechanisms that underlie vertebrate gastrulation movements.
Human embryonic stem cells (hESC) that differentiate into all three primordial germ layers have been established. Differentiation of these cells into desirable lineages offers hope for future transplantation therapies. Currently, hESC lines are derived from the inner cell mass (ICM) of blastocysts, leading to destruction of the embryo, and thus the process is ethically controversial. Successful attempts at deriving hESC lines from blastomeres without destruction of the ensuing embryo have not been reported. One or two blastomeres are routinely biopsied from 8-cell embryos for preimplantation genetic diagnosis. In this study it was therefore attempted to derive hESC lines from paired blastomeres. Of 66 pairs of 8-cell stage blastomeres, four pairs produced two morula and two blastocyst-like structures. When plated on mitomycin-C-treated mouse embryonic fibroblasts, one morula and one blastocyst-like structure separately produced small colonies containing hESC-like cells with prominent nucleoli and high nuclear-cytoplasmic ratios. When these colonies were detached and plated onto fresh feeders, there was no further colony formation or ensuing hESC lines. The results showed that it might not be possible to derive hESC lines directly from paired blastomeres. A minimum number of blastomeres in close contact with one another may be required to successfully generate an hESC line as blastomeres, like ICM and hESC cells, may be 'social' cells.
In September 2003, legislation approved in Denmark legalized work on surplus human embryos from IVF for clinical purposes to establish human embryonic stem (ES) cell cultures. The aim of this study was to establish such stem cell lines. Fresh surplus embryos were donated after informed consent from the donors. Embryos were cultured into blastocysts and using the immunosurgery procedure, inner cell masses were isolated and cultured on irradiated human foreskin fibroblasts in KnockOut D-MEM supplemented with KnockOut Serum Replacement, bFGF, and LIF. Within a period of 12 months, 198 embryos were donated. Four isolated inner cell masses developed into putative ES cell lines, CLS1, CLS2, CLS3, CLS4, which have now been continuously cultured for eight months, corresponding to 30 passages. These cells expressed markers for undifferentiated human ES cells: stage-specific embryonic antigen-4, tumour-related antigen (TRA)-1-60, TRA-1-81, OCT4, NANOG, SOX2, and FGF4. The cells expressed high levels of telomerase activity, had a normal karyotype, and have been successfully cryopreserved and thawed. Finally, the cells displayed the potential to differentiate in vitro into cell types originating from all three germ layers. It is thought that the cell lines described in this study are the first human ES cells established in Denmark.
A total of 184 human embryos, frozen for >5 years, were donated; informed consent was obtained according to Spanish law 45/2003. Survival rate was 40% and three out of 24 blastocysts (12.5%) developed into putative hESC lines, named VAL-3, VAL-4, and VAL-5. The derivation process was performed on microbiologically tested and irradiated human foreskin fibroblasts and designed to minimize contact with xeno-components in knockout DMEM supplemented with knockout serum replacement, and basic fibroblast growth factor. Fingerprinting and HLA typing of the cell lines allowed their identification and traceability. Karyotype was normal for VAL-3 (46XY), VAL-4 (46XX) and VAL-5 (46XX). All three hESC lines expressed specific markers for non-differentiation (Nanog, stage-specific embryonic antigen-4 [SSEA-4], tumour-related antigen [TRA]-1-60, and TRA-1-81) and were negative for SSEA-1. RT-PCR further demonstrated the expression of Oct-4, Sox2, Rex-1, Nanog, Cripto, Thy-1, and Lefty-A. Furthermore, they were found to be negative for classical differentiation markers such as neurofilament heavy chain (ectoderm), renin (mesoderm), and amylase (endoderm). All three cell lines displayed high levels of telomerase activity, and were shown to successfully overcome cryopreservation and thawing. Finally, these three new hESC lines have demonstrated the potential to differentiate in vitro and in vivo (teratoma formation) into cell types originating from all three germ layers.
Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass of the early preimplantation embryo. An efficient strategy for stable genetic modification of hES cells may be highly valuable for manipulating the cells in vitro and may promote the study of hES cell biology, human embryogenesis, and the development of cell-based therapies. Here, we demonstrate that vectors derived from self-inactivating (SIN) human immunodeficiency virus type 1 (HIV-1) are efficient tools for stable genetic modification of hES cells. Transduction of hES cells by a modified vector derived from SIN HIV-1 and containing the woodchuck hepatitis regulatory element (WPRE) and the central polypurine tract (cPPT) sequence facilitated stable transgene expression during prolonged (38 weeks) undifferentiated proliferation in vitro. Southern blot analysis revealed that the viral vector had integrated into the host cells' DNA. Transgene expression was maintained throughout differentiation into progeny of all three germ layers both in vitro and in vivo in teratomas. Thus, the transduced hES cells retained the capability for self-renewal and their pluripotent potential. Genetic modification of hES cells by lentiviral vectors provides a powerful tool for basic and applied research in the area of human ES cells.
Resemblance between animal taxa may be due to convergence rather than to recent common ancestry. Constraints on biological materials and adaptation to particular habits or habitats will produce widespread convergence. How may we distinguish the two causes of resemblance? The relationship between convergence and taxonomy is discussed, demonstrating that the choice of taxonomic method will itself determine the extent to which convergence is perceived. In particular, cladistic analysis based on parsimony will tend to minimise and thus conceal convergence: neither the resulting cladogram nor a consistency index derived from it can be used to assess the prevalence of convergence. With any taxonomic system, there can be no substitute for evaluation of the morphological characters used. Complementary use of molecular characters shows promise: we wait further understanding of constraints in genetic evolution and of the possibilities of convergence at this level also. These general principles are illustrated with a range of examples from within and between invertebrate phyla: the phylogeny of Cnidaria and Platyhelminthes cannot be traced with certainty, but where the fossil record allows clear rooting, as for the echinoderms and in particular the echinoids, combination of morphological and molecular methods has made much progress. Sub-groups within a phylum, for example opisthobranch molluscs and the dipteran Phoridae, may show an uncontested phylogeny, and here studies have precisely identified convergence and shown that it may be the commoner cause of resemblance. Adaptation to exacting environments shown by terrestial and freshwater nemertines may also result in a predominance of convergent resemblance. Traditional grouping of phyla breaks down on re-examination of supposedly key characters, such as segmentation, body cavities, germ layers and symmetry, each of which must have had multiple origins: nor are developmental stages (especially not larvae) a reliable guide to relationships. Demarcation of phyla may be difficult, as with arthropods, and location of phyla is even more difficult, due to their early and rapid radiation. Over-simplified definition of characters has bedevilled invertebrate classification and the use of molecular data has not yet resolved the major controversies. The question "How common is convergence?' remains unanswered and may be unanswerable. Our examples indicate that even the minimum detectable levels of convergence are often high, and we conclude that at all levels convergence has been greatly underestimated.
Teratomas are congenital tumours arising from pluripotent cells and usually consisting of tissue arising from all 3 embryonic germ layers. Teratomas are quite uncommon in head and neck sites and to date only 150 such cases have been reported. A rare case of teratoma of the mastoid region in a newborn is reported. The method of diagnosis and treatment are discussed and the literature is reviewed.
Embryonic stem cells (ESCs) are derived from the inner cell mass of the preimplantation stage embryo and are capable of prolonged symmetrical self-renewal (both daughter cells remain escs) as well as differentiation into derivatives of all three embryonic germ layers. ESCs therefore have the potential to provide an unlimited supply of transplantable cells to replace or regenerate damaged or diseased tissues. However, several barriers must be overcome before successful clinical trials are possible: for example, pure populations of the desired cell type need to be selected and expanded in clinically relevant numbers, and a method for preventing immunological rejection of the transplanted cells without long-term immunosuppressive therapy is also required. In this review, we highlight recent developments in human ESC derivation and expansion, outline current understanding of the signalling pathways underlying stem cell renewal, and discuss challenging problems related to the selective differentiation and immune properties of human ESCs.
Embryonic stem (ES) cells provide a unique tool for cell-mediated gene transfer and targeted gene mutations due to the possibility of in vitro selection of desired genotypes. When selected cells contribute to the germ line in chimaeric embryos, transgenic animals may be generated with modified genetic traits. Though the ES cell approach has up to now been limited to mice, there is an increasing interest to develop this technology in both model and commercial fish species, with so far promising results in the medaka and zebrafish. In this study, we present evidence regarding a long-term stable cell line (SaBE-1c), derived from embryonic cells of the aquaculture marine fish Sparus aurata which has been characterized for (i) cell proliferation, (ii) chromosome complement, (iii) molecular markers, and (iv) in vitro tests of pluripotency by alkaline phosphatase (AP) staining, telomerase activity, and induced cell differentiation. These cells have proved their pluripotent capacities by in vitro tests. Furthermore, we have demonstrated their ability to produce chimaeras and to contribute to the formation of tissues from all three embryonic germ layers. These features suggest that SaBE-1c cells have the potential for multiple applications for the ES technology in fish, with the added value of originating from an economically important species.
Terminally differentiated adult cardiomyocytes have limited regenerative capacity and therefore any significant cell loss may result in the development of progressive heart failure. Cell replacement therapy is a promising new approach for myocardial repair but has been limited by the paucity of cell sources for functional human cardiomyocytes. The recent establishment of the human pluripotent embryonic stem (ES) cell lines may present a novel solution for this cell-sourcing problem. The ES lines were derived from human blastocysts and were shown to be capable of continuous undifferentiated proliferation, in vitro, while retaining the capability to form derivatives of all three germ layers. More recently, a reproducible cardiomyocyte differentiation system was established using these unique cells. The current review describes the derivation and properties of human ES cells and the characteristics of the cardiomyocytes derived using this unique differentiating system. The possible applications in several research and clinical areas are discussed as well as the steps required to fully harness the potential of this new technology in the fields of myocardial cell replacement and tissue engineering.
Interphase fluorescent studies of X chromosome aneuploidy in cultured and uncultured blood lymphocytes and oral mucosa epithelial cells using X centromere-specific DNA probe in addition to standard karyotype analysis were performed in 50 females with a clinical suspicion of Turner syndrome. All the patients were previously screened for the presence of 'hidden' Y chromosome mosaicism, using the primers DYZ3 and DYZ. The use of fluorescence in situ hybridization (FISH) analysis of interphase nuclei of tissues from different germ layers (lymphocytes from mesoderm and buccal epithelial cells from ectoderm) improves the accuracy of detection of low-level mosaicism. FISH studies on interphase nuclei revealed that 29% of patients with a pure form of monosomy X detected by metaphase analysis are, in fact, mosaics. The level of cells with the normal chromosomal constitution in lymphocytes of these cases as a rule was low, ranging from 3 to 18%, with an average of 7%. Two false-positive cases and one false-negative case of X monosomy mosaicism determined by standard cytogenetic approach were detected using FISH analysis. The majority of patients (92%) with mosaic form of Turner syndrome have considerable tissue-specific differences in levels of X aneuploidy. Our data indicate that in cases when mosaic aneuploidy with low-level frequency is questionable (approximately 10% and lower), the results of standard metaphase analysis should be supplemented with additional FISH studies of interphase nuclei. Tissue-specific differences in contents of different cell lines in the same patients point to the necessity of studying more than one tissue from each patient.
The segmented or metameric aspect is a basic characteristic of many animal species ranging from invertebrates to man. Body segmentation usually corresponds to a repetition, along the anteroposterior (AP) axis, of similar structures consisting of derivatives from the three embryonic germ layers. In humans, segmentation is most obvious at the level of the vertebral column and its associated muscles, and also in the peripheral nervous system (PNS). Functionally, segmentation is critical to ensure the movements of a rod-like structure, such as the vertebral column. The segmented distribution of the vertebrae derives from the earlier metameric pattern of the embryonic somites. Recent evidence from work performed in fish, chick and mouse embryos indicates that segmentation of the embryonic body relies on a molecular oscillator called the segmentation clock, which requires Notch signaling for its proper functioning. In humans, mutations in genes required for oscillation, such as Delta-like 3 (DLL3), result in abnormal segmentation of the vertebral column, as found in spondylocostal dysostosis syndrome, suggesting that the segmentation clock also acts during human embryonic development.
Stem cell systems represent an effective and powerful approach for tissue development and regeneration of diverse tissue types. Common and defining features of these exceptional cells are the capacity for self-renewal and the potential for differentiation into multiple mature cell types. Recently, surprising new observations have indicated that stem cells isolated from one adult tissue can also give rise to mature cells of other cell lineages, irrespective of classical germ layer designations. This discovery has resulted in quantum leaps in both scientific knowledge and the potential applications of stem cells. The new findings contradict central dogmas of commitment and differentiation of stem and progenitor cells. However, the true potential of somatic stem cells is just emerging and the new findings have to be defined more fully and integrated into a unifying model of stem cell potential and behavior. Here we analyze the developmental potential of hematopoietic stem cells of mouse and man following their injection into the murine preimplantation blastocyst, an environment that allows the development of all cell lineages. In addition, we discuss the emerging lines of evidence of the developmental plasticity of hematopoietic and other somatic stem cells and consider how cellular memory of transcriptional states is established and may be potentially involved in this phenomenon.