Without epithelial–mesenchymal transitions, in which polarized epithelial cells are converted into motile cells, multicellular organisms would be incapable of getting past the blastula stage of embryonic development. However, this important developmental programme has a more sinister role in tumour progression. Epithelial–mesenchymal transition provides a new basis for understanding the progression of carcinoma towards dedifferentiated and more malignant states.

Epithelial–mesenchymal transitions in tumour progression

We describe the derivation of pluripotent embryonic stem (ES) cells from human blastocysts. Two diploid ES cell lines have been cultivated in vitro for extended periods while maintaining expression of markers characteristic of pluripotent primate cells. Human ES cells express the transcription factor Oct-4, essential for development of pluripotential cells in the mouse. When grafted into SCID mice, both lines give rise to teratomas containing derivatives of all three embryonic germ layers. Both cell lines differentiate in vitro into extraembryonic and somatic cell lineages. Neural progenitor cells may be isolated from differentiating ES cell cultures and induced to form mature neurons. Embryonic stem cells provide a model to study early human embryology, an investigational tool for discovery of novel growth factors and medicines, and a potential source of cells for use in transplantation therapy.

Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro.

The Snail superfamily of zinc-finger transcription factors is involved in processes that imply pronounced cell movements, both during embryonic development and in the acquisition of invasive and migratory properties during tumour progression. Different family members have also been implicated in the signalling cascade that confers left–right identity, as well as in the formation of appendages, neural differentiation, cell division and cell survival.

The snail superfamily of zinc-finger transcription factors.

Before gastrulation, the mouse embryo consists of three distinct cell lineages which were established in the blastocyst during the peri-implantation period, that is, epiblast, extraembryonic endoderm, and trophectoderm. The epiblast, from which the entire fetus will form, as well as the extraembryonic mesoderm and amnion ectoderm, is a cup-shaped epithelium apposed on its open end to the extraembryonic ectoderm, a trophectoderm derivative. Both epiblast and extraembryonic ectoderm are covered by visceral endoderm, which is part of the extraembryonic endoderm lineage (Hogan et al. 1994).

The primordial germ cells (PGCs) of the mouse embryo are derived from part of the population of epiblast cells that will give rise mainly to the extraembryonic mesoderm. Precursors of the PGCs are located before gastrulation in the extreme proximal region of the epiblast adjacent to the extraembryonic ectoderm, and have descendants not only in the germ line, but also in extraembryonic structures, that is, the allantois, blood islands, and yolk sac mesoderm, as well as both layers of the amnion. At embryonic day (E) 6.0, these precursors lie scattered in a ring that extends up to three cell diameters from the junction with the extraembryonic ectoderm (Lawson and Hage 1994). Early in gastrulation, they converge toward the primitive streak in the posterior of the embryo and translocate through it. Allocation to the germ cell lineage is thought to occur in ∼45 cells around E7.2, after the precursors have passed through the streak and have come to reside in the extraembryonic mesoderm (Lawson and Hage 1994). This is about the time when the putative PGCs can first be identified morphologically in a cluster posterior to the primitive streak in a position that will later become the base of the allantois (Ginsburg et al. 1990). PGCs stain strongly in a characteristic pattern for alkaline phosphatase (AP) activity (Chiquoine 1954), which by this stage is due to tissue nonspecific AP (Hahnel et al. 1990; MacGregor et al. 1995). The PGCs continue to express AP during their proliferation in the developing hindgut and migration into the genital ridges (for review, see Buehr 1997).

Transplantation studies have shown that genetically marked distal epiblast cells from pre- and early-primitive streak-stage embryos, which would normally contribute to neuroectoderm and never to the PGCs, can give rise to PGCs and extraembryonic mesoderm when grafted to the proximal epiblast (Tam and Zhou 1996). These results raise the possibility that PGC precursors are induced by extracellular factors and/or cell interactions present locally at the junction between the extraembryonic ectoderm and epiblast.

Candidate genes encoding putative germ cell precursor inducing factors are predicted to be expressed in the mouse embryo before and during gastrulation. One such factor is Bone Morphogenetic Protein 4 (Bmp4), a member of the TGFβ superfamily of intercellular signaling proteins (Hogan 1996; Waldrip et al. 1998). Most mouse embryos homozygous for a null mutation in Bmp4 die around gastrulation (∼E6.5) (Winnier et al. 1995). On some genetic backgrounds, however, a proportion of the mutant embryos survive until the early somite stage and show severe defects, particularly in the extraembryonic mesoderm (Winnier et al. 1995). In this paper, we exploit this late phenotype to show that PGC formation absolutely requires Bmp4 signaling. In addition, the size of the founding population of PGCs is significantly reduced in heterozygous mutant embryos. By using a Bmp4–lacZ reporter allele, we have definitively localized Bmp4 expression before gastrulation in the extraembryonic ectoderm and in mid- to late- primitive streak stage embryos in the extraembryonic mesoderm. Thus, Bmp4 is expressed at the right time and in the right place to play a role both in the quantitative induction of PGC precursors in the proximal epiblast and in their allocation to the germ cell lineage in the extraembryonic mesoderm. Furthermore, by analyzing genetic chimeras, we have clearly established a role for Bmp4 in the induction of PGC precursors and demonstrate for the first time that a secreted signal from the extraembryonic ectoderm is required for the normal development of the epiblast.

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Bmp4 is required for the generation of primordial germ cells in the mouse embryo.

The derivation of neural progenitor cells from human embryonic stem (ES) cells is of value both in the study of early human neurogenesis and in the creation of an unlimited source of donor cells for neural transplantation therapy. Here we report the generation of enriched and expandable preparations of proliferating neural progenitors from human ES cells. The neural progenitors could differentiate in vitro into the three neural lineages--astrocytes, oligodendrocytes, and mature neurons. When human neural progenitors were transplanted into the ventricles of newborn mouse brains, they incorporated in large numbers into the host brain parenchyma, demonstrated widespread distribution, and differentiated into progeny of the three neural lineages. The transplanted cells migrated along established brain migratory tracks in the host brain and differentiated in a region-specific manner, indicating that they could respond to local cues and participate in the processes of host brain development. Our observations set the stage for future developments that may allow the use of human ES cells for the treatment of neurological disorders.

Neural progenitors from human embryonic stem cells.

We describe isolation and characterization of the bovine ortholog of POU5F1 (bPOU5F1) encoding octamer-binding transcription factor-4 (Oct-4). The organization of bPOU5F1 is similar to its human and murine orthologs, and it shares 90.6% and 81.7% overall identity at the protein level, respectively. Transient transfection of luciferase reporter constructs in murine P19 embryonal carcinoma cells demonstrated that bPOU5F1 has a functional promoter and contains two enhancer elements, of which one is repressed by retinoic acid. bPOU5F1 was mapped to the major histocompatibility complex on chromosome 23. bPOU5F1 mRNA was detected by nested reverse transcriptionpolymerase chain reaction in immature oocytes and in in vitroproduced preattachment-stage embryos. Oct-4 in oocytes and in vitro-produced preattachment-stage embryos was demonstrated by indirect immunofluorescence. Confocal laser scanning microscopy revealed Oct-4 in both the inner cell mass and trophoblast cells of the blastocyst until Day 10 of development. Immunofluorescence performed on the outgrowths formed at Day 13 postfertilization from in vitro-produced Day 8 blastocysts showed Oct-4 staining in all cells. This expression pattern suggests that bPOU5F1 acts early in bovine embryonic development but that its expression is not restricted to pluripotent cells of the blastocyst.

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Molecular Cloning, Genetic Mapping, and Developmental Expression of Bovine POU5F1

Aim: Determination of the phenotype of adult human atrial and ventricular myocytes based on gene expression and morphology. Methods: Atrial and ventricular cardiomyocytes were obtained from patients undergoing cardiac surgery using a modified isolation procedure. Myocytes were isolated and cultured with or without serum. The relative cell attachment promoting efficiency of several reagents was evaluated and compared. Morphological changes during long-term culture were assessed with phase contrast microscopy, morphometric analysis and immunocytochemistry or RT-PCR of sarcomeric markers including α-actinin, myosin light chain-2 (MLC-2) and the adhesion molecule, cadherin. Results: The isolation method produced viable rod-shaped atrial (16.6±6.0%, mean±S.E.; n = 5) and ventricular cells (22.4±8.0%, mean±S.E.; n = 5) in addition to significant numbers of apoptotic and necrotic cells. Cell dedifferentiation was characterized by the loss of sarcomeric structure, condensation and extrusion of sarcomeric proteins. Cells cultured with low serum recovered and assumed a flattened, spread form with two distinct morphologies apparent. Type I cells were large, had extensive sarcolemmal spreading, with stress fibers and nascent myofibrils, whilst type II cells appeared smaller, with more mature myofibril organisation and focal adhesions. Conclusion: Characterization of the redifferentiation capabilities of cultured adult cardiac myocytes in culture, provides an important system for comparing cardiomyocytes differentiating from human stem cells and provides the basis for an in vitro transplantation model to study interaction and communication between primary adult and stem cell-derived cardiomyocytes.

The human adult cardiomyocyte phenotype.

We have analysed the function of transforming growth factor beta (TGF-beta) in yolk sac development in mice by generating somatic chimaeras in which the extraembryonic mesoderm, which gives rise to the endothelial and haematopoietic cells of the yolk sac vasculature, is derived from embryonic stem (ES) cells. The ES cells were stably transfected and express either the full-length type II binding receptor or a kinase-deficient mutant of this receptor. Examination of yolk sacs from chimaeras between E8.5 and 9.5, and analysis of marker expression in embryoid bodies from these mutant ES cell lines in prolonged suspension culture demonstrated that (1) a major function of TGF-beta in yolk sac mesoderm is to regulate production and deposition of fibronectin in the extracellular matrix that maintains yolk sac integrity, (2) TGF-beta signalling is not required for differentiation of extraembryonic mesoderm into endothelial cells but is necessary for their subsequent organisation into robust vessels, and (3) TGF-beta signalling must be tightly regulated for the differentiation of primitive haematopoietic cells to take place normally. Together, these results show that defective TGF-beta signalling in the extraembryonic mesoderm alone is sufficient to account for the extraembryonic phenotype reported previously in TGF-beta1(−/−) mice (Dickson, M. C., Martin, J. S., Cousins, F. M., Kulkarni, A. B., Karlsson, S. and Akhurst, R. J. (1995) Development 121, 1845–1854).

Transforming growth factor-beta signalling in extraembryonic mesoderm is required for yolk sac vasculogenesis in mice.

Gastrulation in rodents is associated with an increase in the rate of growth and with the start of differentiation within the embryo proper. In an effort to understand the role played by the cell cycle control in these processes, expression of cyclin D1, D2, and D3--three major positive regulators of the G1/S transition--has been investigated by in situ hybrization and RT-PCR. Cyclin D1 and D2 transcripts are first detected in the epiblast at gastrulation, when a proliferative burst occurs, and subsequently in its differentiated derivatives within the embryo proper, indicating that activation of their expression takes place prior to the differentiation of epiblast progenitors. In contrast, cyclin D3 transcript is undetectable in the epiblast itself and its expression is activated exclusively in extraembryonic tissues of both epiblast and trophoblast origin. During neurulation, expression of each cyclin D RNA is dynamically regulated along the anterior-posterior axis. In the hindbrain, cyclin D1 and D2 show distinct segment-specific restricted expression and this pattern is conserved between mouse and chick. These results strongly suggest that D-type cyclins act as developmental regulators.

M.A. van Rooijen

Papers

Molecular Cloning, Genetic Mapping, and Developmental Expression of Bovine POU5F1

The human adult cardiomyocyte phenotype.

Transforming growth factor-beta signalling in extraembryonic mesoderm is required for yolk sac vasculogenesis in mice.

G1‐phase regulators, cyclin D1, cyclin D2, and cyclin D3: Up‐regulation at gastrulation and dynamic expression during neurulation

Cell cycle analysis during retinoic acid induced differentiation of a human embryonal carcinoma-derived cell line