The gastrointestinal epithelium is unique in that cell proliferation, <em>differentiation</em>, and apoptosis occur in an orderly fashion along the crypt-villus axis. The intestinal crypt is mainly a proliferative compartment, is monoclonal and is maintained by stem cells. The villus represents the differentiated compartment, and is polyclonal as it receives cells from multiple crypts. In the small intestine, cell migration begins near the base of the crypt, and cells migrate from here emerging onto the villi. The basal crypt cells at position 5 are candidate stem cells. As the function of stem cells is to maintain the integrity of the intestinal epithelium, it must self-renew, proliferate, and differentiate within a protective niche. This niche is made up of proliferating and differentiating epithelial cells and surrounding mesenchymal cells. These mesenchymal cells promote the epithelial- mesenchymal crosstalk required to maintain the niche. A stochastic model of cell division has been proposed to explain how a single common ancestral stem cell exists from which all stem cells in a niche are descended. Our group has argued that these crypts then clonally expand by crypt fission, forming two daughters' crypts, and that this is the mechanism by which mutated stem cells or even cancer stem cell clones expand in the colon and in the entire gastrointestinal tract. Until recently, the <em>differentiation</em> potential of stem cells into adult tissues has been thought to be limited to cell lineages in the organ from which they were derived. Bone marrow cells are rare among adult stem cells regarding their abundance and role in the continuous, lifelong, physiological replenishment of circulating cells. In human and mice experiments, we have shown that bone marrow can contribute to the regeneration of intestinal myofibroblasts and thereby after epithelium following damage, through replacing the cells, which maintain the stem cells niche. Little is known about the markers characterizing the stem and transit amplifying populations of the gastrointestinal tract, although musashi-<em>1</em> and hairy and enhancer of split homolog-<em>1</em> have been proposed. As the mammalian gastrointestinal tract develops from the <em>embryonic</em> gut, it is made up of an endodermally-derived epithelium surrounded by cells of mesoderm origin. Cell signaling between these two tissue layers plays a critical role in coordinating patterning and organogenesis of the gut and its derivatives. Many lines of evidence have revealed that Wnt signaling is the most dominant force in controlling cell proliferation, <em>differentiation</em>, and apoptosis along the crypt-villus axis. We have found Wnt messenger RNAs expression in intestinal subepithelial myofibroblasts and frizzled messenger RNAs expression in both myofibroblasts and crypt epithelium. Moreover, there are many other <em>factors</em>, for example, bone morphogenetic protein, homeobox, forkhead, hedgehog, homeodomain, and platelet-derived <em>growth</em> <em>factor</em> that are also important to stem cell signaling in the gastrointestinal tract.

The epithelial-to-mesenchymal transition (EMT) is an <em>embryonic</em> process that becomes latent in most normal adult tissues. Recently, we have shown that induction of EMT endows breast epithelial cells with stem cell traits. In this report, we have further characterized the EMT-derived cells and shown that these cells are similar to mesenchymal stem cells (MSCs) with the capacity to differentiate into multiple tissue lineages. For this purpose, we induced EMT by ectopic expression of Twist, Snail, or transforming <em>growth</em> <em>factor</em>-beta in immortalized human mammary epithelial cells. We found that the EMT-derived cells and MSCs share many properties including the antigenic profile typical of MSCs, that is, CD44(+), CD24(-), and CD45(-). Conversely, MSCs express EMT-associated genes, such as Twist, Snail, and mesenchyme forkhead <em>1</em> (FOXC2). Interestingly, CD<em>1</em>40b (platelet-derived <em>growth</em> <em>factor</em> receptor-beta), a marker for naive MSCs, is exclusively expressed in EMT-derived cells and not in their epithelial counterparts. Moreover, functional analyses revealed that EMT-derived cells but not the control cells can differentiate into alizarin red S-positive mature osteoblasts, oil red O-positive adipocytes and alcian blue-positive chondrocytes similar to MSCs. We also observed that EMT-derived cells but not the control cells invade and migrate towards MDA-MB-23<em>1</em> breast cancer cells similar to MSCs. In vivo wound homing assays in nude mice revealed that the EMT-derived cells home to wound sites similar to MSCs. In conclusion, we have demonstrated that the EMT-derived cells are similar to MSCs in gene expression, multilineage <em>differentiation</em>, and ability to migrate towards tumor cells and wound sites.

Vascular endothelial <em>growth</em> <em>factor</em> (VEGF)-mediated angiogenesis is an important part of bone formation. To clarify the role of VEGF isoforms in endochondral bone formation, we examined long bone development in mice expressing exclusively the VEGF<em>1</em>20 isoform (VEGF<em>1</em>20/<em>1</em>20 mice). Neonatal VEGF<em>1</em>20/<em>1</em>20 long bones showed a completely disturbed vascular pattern, concomitant with a 35% decrease in trabecular bone volume, reduced bone <em>growth</em> and a 34% enlargement of the hypertrophic chondrocyte zone of the <em>growth</em> plate. Surprisingly, <em>embryonic</em> hindlimbs at a stage preceding capillary invasion exhibited a delay in bone collar formation and hypertrophic cartilage calcification. Expression levels of marker genes of osteoblast and hypertrophic chondrocyte <em>differentiation</em> were significantly decreased in VEGF<em>1</em>20/<em>1</em>20 bones. Furthermore, inhibition of all VEGF isoforms in cultures of <em>embryonic</em> cartilaginous metatarsals, through the administration of a soluble receptor chimeric protein (mFlt-<em>1</em>/Fc), retarded the onset and progression of ossification, suggesting that osteoblast and/or hypertrophic chondrocyte development were impaired. The initial invasion by osteoclasts and endothelial cells into VEGF<em>1</em>20/<em>1</em>20 bones was retarded, associated with decreased expression of matrix metalloproteinase-9. Our findings indicate that expression of VEGF<em>1</em>64 and/or VEGF<em>1</em>88 is important for normal endochondral bone development, not only to mediate bone vascularization but also to allow normal <em>differentiation</em> of hypertrophic chondrocytes, osteoblasts, endothelial cells and osteoclasts.

The mechanisms involved in the regulation of vasculogenesis still remain unclear in mammals. Totipotent <em>embryonic</em> stem (ES) cells may represent a suitable in vitro model to study molecular events involved in vascular development. In this study, we followed the expression kinetics of a relatively large set of endothelial-specific markers in ES-derived embryoid bodies (EBs). Results of both reverse transcription-polymerase chain reaction and/or immunofluorescence analysis show that a spontaneous endothelial <em>differentiation</em> occurs during EBs development. ES-derived endothelial cells express a full range of cell lineage-specific markers: platelet endothelial cell adhesion molecule (PECAM), Flk-<em>1</em>, tie-<em>1</em>, tie-2, vascular endothelial (VE) cadherin, MECA-32, and MEC-<em>1</em>4.7. Analysis of the kinetics of endothelial marker expression allows the <em>distinction</em> of successive maturation steps. Flk-<em>1</em> was the first to be detected; its mRNA is apparent from day 3 of <em>differentiation</em>. PECAM and tie-2 mRNAs were found to be expressed only from day 4, whereas VE-cadherin and tie-<em>1</em> mRNAs cannot be detected before day 5. Immunofluorescence stainings of EBs with antibodies directed against Flk-<em>1</em>, PECAM, VE-cadherin, MECA-32, and MEC-<em>1</em>4.7 confirmed that the expression of these antigens occurs at different steps of endothelial cell <em>differentiation</em>. The addition of an angiogenic <em>growth</em> <em>factor</em> mixture including erythropoietin, interleukin-6, fibroblast <em>growth</em> <em>factor</em> 2, and vascular endothelial <em>growth</em> <em>factor</em> in the EB culture medium significantly increased the development of primitive vascular-like structures within EBs. These results indicate that this in vitro system contains a large part of the endothelial cell <em>differentiation</em> program and constitutes a suitable model to study the molecular mechanisms involved in vasculogenesis.

GATA-<em>1</em> is a zinc-finger transcription <em>factor</em> believed to play an important role in gene regulation during the development of erythroid cells, megakaryocytes and mast cells. Other members of the GATA family, which can bind to the same DNA sequence motif, are co-expressed in several of these hemopoietic lineages, raising the possibility of overlap in function. To examine the specific roles of GATA-<em>1</em> in hematopoietic cell <em>differentiation</em>, we have tested the ability of <em>embryonic</em> stem cells, carrying a targeted mutation in the X-linked GATA-<em>1</em> gene, to contribute to various blood cell types when used to produce chimeric embryos or mice. Previously, we reported that GATA-<em>1</em>- mutant cells failed to contribute to the mature red blood cell population, indicating a requirement for this <em>factor</em> at some point in the erythroid lineage (L. Pevny et al., (<em>1</em>99<em>1</em>) Nature 349, 257-260). In this study, we have used in vitro colony assays to identify the stage at which mutant erythroid cells are affected, and to examine the requirement for GATA-<em>1</em> in other lineages. We found that the development of erythroid progenitors in <em>embryonic</em> yolk sacs was unaffected by the mutation, but that the cells failed to mature beyond the proerythroblast stage, an early point in terminal <em>differentiation</em>. GATA-<em>1</em>- colonies contained phenotypically normal macrophages, neutrophils and megakaryocytes, indicating that GATA-<em>1</em> is not required for the in vitro <em>differentiation</em> of cells in these lineages. GATA-<em>1</em>- megakaryocytes were abnormally abundant in chimeric fetal livers, suggesting an alteration in the kinetics of their formation or turnover. The lack of a block in terminal megakaryocyte <em>differentiation</em> was shown by the in vivo production of platelets expressing the ES cell-derived GPI-<em>1</em>C isozyme. The role of GATA-<em>1</em> in mast cell <em>differentiation</em> was examined by the isolation of clonal mast cell cultures from chimeric fetal livers. Mutant and wild-type mast cells displayed similar <em>growth</em> and histochemical staining properties after culture under conditions that promote the <em>differentiation</em> of cells resembling mucosal or serosal mast cells. Thus, the mast and megakaryocyte lineages, in which GATA-<em>1</em> and GATA-2 are co-expressed, can complete their maturation in the absence of GATA-<em>1</em>, while erythroid cells, in which GATA-<em>1</em> is the predominant GATA <em>factor</em>, are blocked at a relatively early stage of maturation.

Neural progenitor cells obtained from the <em>embryonic</em> human forebrain were expanded up to <em>1</em>0(7)-fold in culture in the presence of epidermal <em>growth</em> <em>factor</em>, basic fibroblast <em>growth</em> <em>factor</em>, and leukemia inhibitory <em>growth</em> <em>factor</em>. When transplanted into neurogenic regions in the adult rat brain, the subventricular zone, and hippocampus, the in vitro propagated cells migrated specifically along the routes normally taken by the endogenous neuronal precursors: along the rostral migratory stream to the ol<em>factor</em>y bulb and within the subgranular zone in the dentate gyrus, and exhibited site-specific neuronal <em>differentiation</em> in the granular and periglomerular layers of the bulb and in the dentate granular cell layer. The cells exhibited substantial migration also within the non-neurogenic region, the striatum, in a seemingly nondirected manner up to approximately <em>1</em>-<em>1</em>.5 mm from the graft core, and showed <em>differentiation</em> into both neuronal and glial phenotypes. Only cells with glial-like features migrated over longer distances within the mature striatum, whereas the cells expressing neuronal phenotypes remained close to the implantation site. The ability of the human neural progenitors to respond in vivo to guidance cues and signals that can direct their <em>differentiation</em> along multiple phenotypic pathways suggests that they can provide a powerful and virtually unlimited source of cells for experimental and clinical transplantation.

We have compared the expression of the genes encoding transforming <em>growth</em> <em>factors</em> beta <em>1</em>, beta 2 and beta 3 during mouse embryogenesis from 9.5 to <em>1</em>6.5 days p.c. using in situ hybridisation to cellular RNAs. Each gene has a different expression pattern, which gives some indication of possible biological function in vivo. All three genes appear to be involved in chondroossification, though each is expressed in a different cell type. Transcripts of each gene are also present in <em>embryonic</em> epithelia. Epithelial expression of TGF beta <em>1</em>, beta 2 and beta 3 RNA is associated with regions of active morphogenesis involving epithelial-mesenchymal interactions. In addition, widespread epithelial expression of TGF beta 2 RNA can be correlated with epithelial <em>differentiation</em> per se. The localisation of TGF beta 2 RNA in neuronal tissue might also be correlated with <em>differentiation</em>. Finally both TGF beta <em>1</em> and beta 2 transcripts are seen in regions actively undergoing cardiac septation and valve formation, suggesting some interaction of these <em>growth</em> <em>factors</em> in this developmental process.

Overexpression of upstream of <em>growth</em> and <em>differentiation</em> <em>factor</em> <em>1</em> (uog<em>1</em>), a mammalian homolog of the yeast longevity assurance gene (LAG<em>1</em>), selectively induces the synthesis of stearoyl-containing sphingolipids in mammalian cells (Venkataraman, K., Riebeling, C., Bodennec, J., Riezman, H., Allegood, J. C., Sullards, M. C., Merrill, A. H. Jr., and Futerman, A. H. (2002) J. Biol. Chem. 277, 35642-35649). Gene data base analysis subsequently revealed a new subfamily of proteins containing the Lag<em>1</em>p motif, previously characterized as translocating chain-associating membrane (TRAM) protein homologs (TRH). We now report that two additional members of this family regulate the synthesis of (dihydro)ceramides with specific fatty acid(s) when overexpressed in human <em>embryonic</em> kidney 293T cells. TRH<em>1</em> or TRH4-overexpression elevated [3H](dihydro)ceramide synthesis from l-[3-3H]serine and the increase was not blocked by the (dihydro)ceramide synthase inhibitor, fumonisin B<em>1</em> (FB<em>1</em>). Analysis of sphingolipids by liquid chromatography-electrospray tandem mass spectrometry revealed that TRH4 overexpression elevated mainly palmitic acid-containing sphingolipids whereas TRH<em>1</em> overexpression increased mainly stearic acid and arachidic acid, which in both cases were further elevated upon incubation with FB<em>1</em>. A similar fatty acid specificity was obtained upon analysis of (dihydro)ceramide synthase activity in vitro using various fatty acyl-CoA substrates, although in a FB<em>1</em>-sensitive manner. Moreover, in homogenates from TRH4-overexpressing cells, sphinganine, rather than sphingosine was the preferred substrate, whereas no preference was seen in homogenates from TRH<em>1</em>-overexpressing cells. These findings lend support to our hypothesis (Venkataraman, K., and Futerman, A. H. (2002) FEBS Lett. 528, 3-4) that Lag<em>1</em>p family members regulate (dihydro)ceramide synthases responsible for production of sphingolipids containing different fatty acids.

During recent years, our understanding of TGFbeta signalling through serine/threonine kinase receptors and Smads has increased enormously. Activation of R-Smads by receptor induced phosphorylation is followed by complex formation with co-Smads and translocation to the nucleus, where the transcription of specific genes is affected and ultimately results in changes in cell behaviour. Experimental analysis primarily of epithelial cells in culture has revealed that a number of members of the TGFbeta family are interchangeable in the effect they have on <em>growth</em> and <em>differentiation</em>. On the other hand, different ligands of the TGFbeta superfamily can result in different responses because of cell type specific expression of other components of the signalling pathway. The relative expression levels of receptors and Smads within the cell is an important determinant of TGFbeta induced responses. Functional analysis of genes in the TGFbeta superfamily signal transduction cascade in vivo in mice either lacking entire genes, or expressing dominant negative forms of particular proteins, are providing profound new insights into the signalling cascades, their interaction and their specificity (Table 3). For example, by phenotypical comparison and intercrossing different heterozygous mutants, it has become clear that nodal, until recently an orphan protein without receptor/signal complex, probably signals through the activin type II receptor, ALK-4 and Smad2 (Nomura and Li, <em>1</em>998; Song et al., <em>1</em>999). Many of the genes of this cascade that have been targeted in the mouse result in early <em>embryonic</em> lethal phenotypes, demonstrating an important function for the BMP and TGFbeta/activin-activated pathways in mesoderm formation and <em>differentiation</em>, but masking a possible role in later events. For example mutations in BMP2 and 4 are lethal at or soon after gastrulation so that their putative role in skeletogenesis cannot be studied in mice lacking these genes. The <em>difference</em> in severity of the phenotypes between ligand, receptor and Smad deficient mice suggest that other receptors and ligands may partially compensate for the loss of one protein. Chimeric analysis provides one tool for analysing later developmental functions. By rescuing the early defects it was demonstrated that TGFbeta family members have an important function in anterior development and left/right asymmetry. Temporal and spatial specific gene targeting will be a powerful tool for analysing the function of TGFbeta family members in for example, bone formation, angiogenesis and carcinogenesis. Isolation of cells from the different gene targeted mice provides a unique source of material to gain more insight in the biochemical mechanisms of specific pathways. For example, use of cells deficient in Smad2 for biochemical and cell biological assays could give a better view of the function of Smad3. Smad3 deficient mice already demonstrate that there is a clear <em>difference</em> between Smad2 and Smad3 during development. Full descriptions of the remaining gene ablation studies of this signal transduction cascade, namely those for ALK-5, BMPR-II and Smad<em>1</em> and -7 are eagerly awaited to complete the puzzle. As more of these superfamily of ligands and their signalling pathways have been functionally dissected, it has become evident that this superfamily of <em>growth</em> <em>factors</em> plays a pivotal role in epiblast formation and gastrulation, signalling from both the epiblast as well as the extra<em>embryonic</em> tissues. Furthermore, it becomes clear that TGFbeta is indeed important for proper vessel formation and that it might use endoglin, as well as ALK-<em>1</em>, ALK-5 and Smad5 to mediate this function. Further analyses of these mice should provide a clearer understanding of the mechanism of TGFbeta action in vascular development and remodelling.

Mitochondrial morphology is crucial for tissue homeostasis, but its role in cell <em>differentiation</em> is unclear. We found that mitochondrial fusion was required for proper cardiomyocyte development. Ablation of mitochondrial fusion proteins Mitofusin <em>1</em> and 2 in the <em>embryonic</em> mouse heart, or gene-trapping of Mitofusin 2 or Optic atrophy <em>1</em> in mouse <em>embryonic</em> stem cells (ESCs), arrested mouse heart development and impaired <em>differentiation</em> of ESCs into cardiomyocytes. Gene expression profiling revealed decreased levels of transcription <em>factors</em> transforming <em>growth</em> <em>factor</em>-β/bone morphogenetic protein, serum response <em>factor</em>, GATA4, and myocyte enhancer <em>factor</em> 2, linked to increased Ca(2+)-dependent calcineurin activity and Notch<em>1</em> signaling that impaired ESC <em>differentiation</em>. Orchestration of cardiomyocyte <em>differentiation</em> by mitochondrial morphology reveals how mitochondria, Ca(2+), and calcineurin interact to regulate Notch<em>1</em> signaling.

Fibroblast <em>growth</em> <em>factors</em> (FGFs) have been implicated in many aspects of cell <em>growth</em> and <em>differentiation</em> both in normal and neoplastic settings. For example, the mouse int-2 gene, which encodes an FGF-related product, is a frequent target of proviral activation in carcinomas induced by mouse mammary tumour virus, but apparently functions at discrete stages of normal <em>embryonic</em> development. Six classes of int-2 messenger RNA have been identified in <em>embryonic</em> cells, each of which is predicted to encode the same 245-amino-acid protein. But all known int-2 transcripts include sequences upstream of the AUG codon presumed to be the initiation codon. Here we report an additional N-terminally extended int-2 gene product initiated at an in-frame CUG codon. In COS-<em>1</em> cells transiently transfected with appropriate int-2 complementary DNAs, the AUG-initiated product is found predominantly in the secretory pathway, whereas the CUG-initiated form is localized to the nucleus. These data indicate that the Int-2 oncoprotein could influence cellular behaviour by two distinct mechanisms.

Transforming <em>growth</em> <em>factor</em>-beta / bone morphogenetic protein (TGFbeta/BMP) signaling has a gradient of effects on cell fate choice in the fetal mouse liver. The molecular mechanism to understand why adjacent cells develop into bile ducts or grow actively as hepatocytes in the ubiquitous presence of both TGFbeta ligands and receptors has been unknown. We hypothesized that microRNAs (miRNAs) might play a role in cell fate decisions in the liver. miRNA profiling during late fetal development in the mouse identified miR-23b cluster miRNAs comprising miR-23b, miR-27b, and miR-24-<em>1</em> and miR-<em>1</em>0a, miR-26a, and miR-30a as up-regulated. In situ hybridization of fetal liver at <em>embryonic</em> day <em>1</em>7.5 of gestation revealed miR-23b cluster expression only in fetal hepatocytes. A complementary (c)DNA microarray approach was used to identify genes with a reciprocal expression pattern to that of miR-23b cluster miRNAs. This approach identified Smads (mothers against decapentaplegic homolog), the key TGFbeta signaling molecules, as putative miR-23b cluster targets. Bioinformatic analysis identified multiple candidate target sites in the 3' UTRs (untranslated regions) of Smads 3, 4, and 5. Dual luciferase reporter assays confirmed down-regulation of constructs containing Smad 3, 4, or 5, 3' UTRs by a mixture of miR-23b cluster mimics. Knockdown of miR-23b miRNAs during hepatocytic <em>differentiation</em> of a fetal liver stem cell line, HBC-3, promoted expression of bile duct genes, in addition to Smads, in these cells. In contrast, ectopic expression of miR-23b mimics during bile duct <em>differentiation</em> of HBC-3 cells blocked the process.

CONCLUSIONS

Our data provide a model in which miR-23b miRNAs repress bile duct gene expression in fetal hepatocytes while promoting their growth by down-regulating Smads and consequently TGFbeta signaling. Concomitantly, low levels of the miR-23b miRNAs are needed in cholangiocytes to allow TGFbeta signaling and bile duct formation.

<em>Growth</em> <em>factor</em>-induced signaling by receptor tyrosine kinases (RTKs) plays a central role in <em>embryonic</em> development and in pathogenesis and, hence, is tightly controlled by several regulatory proteins. Recently, Sprouty, an inhibitor of Drosophila development-associated RTK signaling, has been discovered. Subsequently, four mammalian Sprouty homologues (Spry-<em>1</em>-4) have been identified. Here, we report the functional characterization of two of them, Spry-<em>1</em> and -2, in endothelial cells. Overexpressed Spry-<em>1</em> and -2 inhibit fibroblast <em>growth</em> <em>factor</em>- and vascular endothelial <em>growth</em> <em>factor</em>-induced proliferation and <em>differentiation</em> by repressing pathways leading to p42/44 mitogen-activating protein (MAP) kinase activation. In contrast, although epidermal <em>growth</em> <em>factor</em>-induced proliferation of endothelial cells was also inhibited by Spry-<em>1</em> and -2, activation of p42/44 MAP kinase was not affected. Biochemical and immunofluorescence analysis of endogenous and overexpressed Spry-<em>1</em> and -2 reveal that both Spry-<em>1</em> and -2 are anchored to membranes by palmitoylation and associate with caveolin-<em>1</em> in perinuclear and vesicular structures. They are phosphorylated on serine residues and, upon <em>growth</em> <em>factor</em> stimulation, a subset is recruited to the leading edge of the plasma membrane. The data indicate that mammalian Spry-<em>1</em> and -2 are membrane-anchored proteins that negatively regulate angiogenesis-associated RTK signaling, possibly in a RTK-specific fashion.

The primary function of the thyroid gland is to metabolize iodide by synthesizing thyroid hormones, which are critical regulators of <em>growth</em>, development and metabolism in almost all tissues. So far, research on thyroid morphogenesis has been missing an efficient stem-cell model system that allows for the in vitro recapitulation of the molecular and morphogenic events regulating thyroid follicular-cell <em>differentiation</em> and subsequent assembly into functional thyroid follicles. Here we report that a transient overexpression of the transcription <em>factors</em> NKX2-<em>1</em> and PAX8 is sufficient to direct mouse <em>embryonic</em> stem-cell <em>differentiation</em> into thyroid follicular cells that organize into three-dimensional follicular structures when treated with thyrotropin. These in vitro-derived follicles showed appreciable iodide organification activity. Importantly, when grafted in vivo into athyroid mice, these follicles rescued thyroid hormone plasma levels and promoted subsequent symptomatic recovery. Thus, mouse <em>embryonic</em> stem cells can be induced to differentiate into thyroid follicular cells in vitro and generate functional thyroid tissue.

Xenopus in vitro studies have implicated both transforming <em>growth</em> <em>factor</em> beta (TGF-beta) and fibroblast <em>growth</em> <em>factor</em> (FGF) families in mesoderm induction. Although members of both families are present during mouse mesoderm formation, there is little evidence for their functional role in mesoderm induction. We show that mouse <em>embryonic</em> stem cells, which resemble primitive ectoderm, can differentiate to mesoderm in vitro in a chemically defined medium (CDM) in the absence of fetal bovine serum. In CDM, this <em>differentiation</em> is responsive to TGF-beta family members in a concentration-dependent manner, with activin A mediating the formation of dorsoanterior-like mesoderm and bone morphogenetic protein 4 mediating the formation of ventral mesoderm, including hematopoietic precursors. These effects are not observed in CDM alone or when TGF-beta <em>1</em>, -beta 2, or -beta 3, acid FGF, or basic FGF is added individually to CDM. In vivo, at day 6.5 of mouse development, activin beta A RNA is detectable in the decidua and bone morphogenetic protein 4 RNA is detectable in the egg cylinder. Together, our data strongly implicate the TGF-beta family in mammalian mesoderm development and hematopoietic cell formation.

Angiogenesis is a fundamental vertebrate developmental process that requires signalling by the secreted protein vascular endothelial <em>growth</em> <em>factor</em>-A (VEGF-A). VEGF-A functions in the development of <em>embryonic</em> structures, during tissue remodelling and for the <em>growth</em> of tumour-induced vasculature. The study of the role of VEGF-A during normal development has been significantly complicated by the dominant, haplo-insufficient nature of VEGF-A-targeted mutations in mice. We have used morpholino-based targeted gene knock-down technology to generate a zebrafish VEGF-A morphant loss of function model. Zebrafish VEGF-A morphant embryos develop with an enlarged pericardium and with major blood vessel deficiencies. Morphological assessment at 2 days of development indicates a nearly complete absence of both axial and intersegmental vasculature, with no or reduced numbers of circulating red blood cells. Molecular analysis using the endothelial markers fli-<em>1</em> and flk-<em>1</em> at <em>1</em> day of development demonstrates a fundamental <em>distinction</em> between VEGF-A requirements for axial and intersegmental vascular structure specification. VEGF-A is not required for the initial establishment of axial vasculature patterning, whereas all development of intersegmental vasculature is dependent on VEGF-A signalling. The zebrafish thus serves as a quality model for the study of conserved vertebrate angiogenesis processes during <em>embryonic</em> development.

Pluripotency is a unique biological state that allows cells to differentiate into any tissue type. Here we describe a candidate pluripotency <em>factor</em>, Ronin, that possesses a THAP domain, which is associated with sequence-specific DNA binding and epigenetic silencing of gene expression. Ronin is expressed primarily during the earliest stages of murine <em>embryonic</em> development, and its deficiency in mice produces periimplantational lethality and defects in the inner cell mass. Conditional knockout of Ronin prevents the <em>growth</em> of ES cells while forced expression of Ronin allows ES cells to proliferate without <em>differentiation</em> under conditions that normally do not promote self-renewal. Ectopic expression also partly compensates for the effects of Oct4 knockdown. We demonstrate that Ronin binds directly to HCF-<em>1</em>, a key transcriptional regulator. Our findings identify Ronin as an essential <em>factor</em> underlying embryogenesis and ES cell pluripotency. Its association with HCF-<em>1</em> suggests an epigenetic mechanism of gene repression in pluripotent cells.

Control of <em>growth</em> and <em>differentiation</em> during mammalian embryogenesis may be regulated by <em>growth</em> <em>factors</em> from <em>embryonic</em> or maternal sources. With the use of single-cell messenger RNA phenotyping, the simultaneous expression of <em>growth</em> <em>factor</em> transcripts in single or small numbers of preimplantation mouse embryos was examined. Transcripts for platelet-derived <em>growth</em> <em>factor</em> A chain (PDGF-A), transforming <em>growth</em> <em>factor</em> (TGF)-alpha, and TGF-beta <em>1</em>, but not for four other <em>growth</em> <em>factors</em>, were found in whole blastocysts. TGF-alpha, TGF-beta <em>1</em>, and PDGF antigens were detected in blastocysts by immunocytochemistry. Both PDGF-A and TGF-alpha were detected as maternal transcripts in the unfertilized ovulated oocyte, and again in blastocysts. TGF-beta <em>1</em> transcripts appeared only after fertilization. The expression of a subset of <em>growth</em> <em>factors</em> in mouse blastocysts suggests a role for these <em>factors</em> in the <em>growth</em> and <em>differentiation</em> of early mammalian embryos.

Acquisition of a cardiac fate by <em>embryonic</em> mesodermal cells is a fundamental step in heart formation. Heart development in frogs and avians requires positive signals from adjacent endoderm, including bone morphogenic proteins, and is antagonized by a second secreted signal, Wnt proteins, from neural tube. By contrast, mechanisms of mesodermal commitment to create heart muscle in mammals are largely unknown. In addition, Wnt-dependent signals can involve either a canonical beta-catenin pathway or other, alternative mediators. Here, we tested the involvement of Wnts and beta-catenin in mammalian cardiac myogenesis by using a pluripotent mouse cell line (P<em>1</em>9CL6) that recapitulates early steps for cardiac specification. In this system, early and late cardiac genes are up-regulated by <em>1</em>% DMSO, and spontaneous beating occurs. Notably, Wnt3A and Wnt8A were induced days before even the earliest cardiogenic transcription <em>factors</em>. DMSO induced biochemical mediators of Wnt signaling (decreased phosphorylation and increased levels of beta-catenin), which were suppressed by Frizzled-8Fc, a soluble Wnt antagonist. DMSO provoked T cell <em>factor</em>-dependent transcriptional activity; thus, induction of Wnt proteins by DMSO was functionally coupled. Frizzled-8Fc inhibited the induction of cardiogenic transcription <em>factors</em>, cardiogenic <em>growth</em> <em>factors</em>, and sarcomeric myosin heavy chains. Likewise, <em>differentiation</em> was blocked by constitutively active glycogen synthase kinase 3beta, an intracellular inhibitor of the Wntbeta-catenin pathway. Conversely, lithium chloride, which inhibits glycogen synthase kinase 3beta, and Wnt3A-conditioned medium up-regulated early cardiac markers and the proportion of differentiated cells. Thus, Wntbeta-catenin signaling is activated at the inception of mammalian cardiac myogenesis and is indispensable for cardiac <em>differentiation</em>, at least in this pluripotent model system.

Totipotent germline blastomeres in Caenorhabditis elegans contain, but do not respond to, <em>factors</em> that promote somatic <em>differentiation</em> in other <em>embryonic</em> cells. Mutations in the maternal gene pie-<em>1</em> result in the germline blastomeres adopting somatic cell fates. Here we show that pie-<em>1</em> encodes a nuclear protein, PIE-<em>1</em>, that is localized to the germline blastomeres throughout early development. During division of each germline blastomere, PIE-<em>1</em> initially associates with both centrosomes of the mitotic spindle. However, PIE-<em>1</em> rapidly disappears from the centrosome destined for the somatic daughter, and persists in the centrosome of the daughter that becomes the next germline blastomere. The PIE-<em>1</em> protein contains potential zinc-finger motifs also found in the mammalian <em>growth</em>-<em>factor</em> response protein TIS-<em>1</em><em>1</em>/NUP475 (refs 4-7). The localization and genetic properties of pie-<em>1</em> provide an example of a repressor-based mechanism for preserving pluripotency within a stem cell lineage.

During early pregnancy, placentation occurs in a relatively hypoxic environment which is essential for appropriate <em>embryonic</em> development. Intervillous blood flow increases at around <em>1</em>0-<em>1</em>2 weeks of gestation and results in exposure of the trophoblast to increased oxygen tension (PO2). Prior to this time, low oxygen appears to prevent trophoblast <em>differentiation</em> towards an invasive phenotype. In other mammalian systems, oxygen tension effects are mediated by hypoxia inducible <em>factor</em>-<em>1</em> (HIF-<em>1</em>). We found that the ontogeny of HIF-<em>1</em>alpha subunit expression during the first trimester of gestation parallels that of transforming <em>growth</em> <em>factor</em>-beta3 (TGFbeta3), an inhibitor of early trophoblast <em>differentiation</em>. Expression of both molecules is high in early pregnancy and falls at around <em>1</em>0 weeks of gestation when placental PO2 levels are believed to increase. Antisense-induced inhibition of HIF-<em>1</em>alpha inhibited the expression of TGFbeta3, and stimulated extravillous trophoblast (EVT) out<em>growth</em> and invasion. Of clinical significance we found that TGFbeta3 expression was increased in pre-eclamptic placentae when compared to age-matched controls. Significantly, inhibition of TGFbeta3 by antisense oligonucleotides or antibodies restored the invasive capability to the trophoblast cells in pre-eclamptic explants. We speculate that if oxygen tension fails to increase, or trophoblasts do not detect this increase, HIF-<em>1</em>alpha and TGFbeta3 expression remain high, resulting in shallow trophoblast invasion and predisposing the pregnancy to pre-eclampsia. Effective fetal-maternal interactions during early placentation are critical for a successful pregnancy. Optimal placental perfusion requires the controlled invasion of trophoblast cells deep into the decidua to the spiral arteries. Trophoblast stem cells, also referred to as cytotrophoblast cells, reside in chorionic villi of two types, floating and anchoring villi. Floating villi, which represent the vast majority of chorionic villi, are bathed in maternal blood and primarily perform gas and nutrient exchange for the developing embryo. During early placentation, cytotrophoblast cells in the floating villi proliferate and differentiate by fusing to form the multinucleate syncytiotrophoblast layer. Cytotrophoblast cells in anchoring villi either fuse to form the syncytiotrophoblast layer, or break through the syncytium at selected sites and form multilayered columns of non-polarized extravillous trophoblast cells, which physically connect the embryo to the uterine wall (Figure <em>1</em>). The extravillous trophoblast cells invade into the uterine wall as far as the first third of the myometrium and its associated spiral arteries, where they disrupt the endothelium and the smooth muscle layer and replace the vascular wall. This results in the conversion of the narrow calibre arteries into distended uteroplacental arteries, thereby increasing blood flow to the placenta and allowing an adequate supply of oxygen and nutrients to the <em>growing</em> fetus. The invasive activity of the extravillous trophoblast cells is at a maximum during the first trimester of gestation, peaking at around <em>1</em>0-<em>1</em>2 weeks and declining thereafter. Insufficient invasion contributes to the development of pre-eclampsia, which often results in fetal intrauterine <em>growth</em> restriction, maternal hypertension and proteinuria. In contrast, unrestricted invasion is associated with premalignant conditions, such as invasive mole, and with malignant choriocarcinoma. Invading trophoblast cells undergo striking and rapid changes in cellular functions that are temporally and spatially regulated along the invasive pathway (Figure <em>1</em>) (Cross, Werb and Fisher, <em>1</em>994. The formation of the anchoring villi is accompanied by changes in synthesis and degradation of extracellular matrix proteins and their receptors, and changes in the spatial distribution of extracellular matrix proteins, as well as changes in the expression of adhesion molecules (Damsky, Fitzgerald and

Recent data have demonstrated that vascular endothelial <em>growth</em> <em>factor</em> (VEGF) is expressed by subsets of neurons, coincident with angiogenesis within the developing cerebral cortex. Here we investigate the characteristics of VEGF expression by neurons and test the hypothesis that VEGF may serve both paracrine and autocrine functions in the developing central nervous system. To begin to address these questions, we assayed expression of VEGF and one of its potential receptors, Flk-<em>1</em> (VEGFR-2), in the <em>embryonic</em> mouse forebrain and <em>embryonic</em> cortical neurons grown in vitro. Both VEGF and Flk-<em>1</em> are present in subsets of post-mitotic neurons in vivo and in vitro. Moreover, VEGF levels are up-regulated in neuronal cultures subjected to hypoxia, consistent with our previous results in vivo. While the abundance of Flk-<em>1</em> is unaffected by hypoxia, the receptor exhibits a higher level of tyrosine phosphorylation, as do downstream signaling kinases, including extracellular signal-regulated protein kinase, p90RSK and STAT3a, demonstrating activation of the VEGF pathway. These same signaling components also exhibited higher tyrosine phosphorylation levels in response to exogenous addition of rVEGFA(<em>1</em>65). This activation was diminished in the presence of specific inhibitors of Flk-<em>1</em> function and agents that sequester VEGF, resulting in a dose-dependent increase in apoptosis in these neuronal cultures. Further, inhibition of MEK resulted in increased apoptosis, while inhibition of phosphatidylinositol 3-kinase had no appreciable affect. In addition to the novel function for VEGF that we describe in neuronal survival, neuronal VEGF also affected the organization and <em>differentiation</em> of brain endothelial cells in a three-dimensional culture paradigm, consistent with its more traditional role as a vascular agent. Thus, our in vitro data support a role for neuronal VEGF in both paracrine and autocrine signaling in the maintenance of neurons and endothelia in the central nervous system.

The isolation and expansion of human neural progenitor cells have important potential clinical applications, because these cells may be used as graft material in cell therapies to regenerate tissue and/or function in patients with central nervous system (CNS) disorders. This paper describes a continuously dividing multipotent population of progenitor cells in the human <em>embryonic</em> forebrain that can be propagated in vitro. These cells can be maintained and expanded using a serum-free defined medium containing basic fibroblast <em>growth</em> <em>factor</em> (bFGF), leukemia inhibitory <em>factor</em> (LIF), and epidermal <em>growth</em> <em>factor</em> (EGF). Using these three <em>factors</em>, the cell cultures expand and remain multipotent for at least <em>1</em> year in vitro. This period of expansion results in a <em>1</em>0(7)-fold increase of this heterogeneous population of cells. Upon <em>differentiation</em>, they form neurons, astrocytes, and oligodendrocytes, the three main phenotypes in the CNS. Moreover, GABA-immunoreactive and tyrosine hydroxylase-immunoreactive neurons can be identified. These results demonstrate the feasibility of long-term in vitro expansion of human neural progenitor cells. The advantages of such a population of neural precursors for allogeneic transplantation include the ability to provide an expandable, well-characterized, defined cell source which can form specific neuronal or glial subtypes.

The longevity assurance gene (LAG<em>1</em>) and its homolog (LAC<em>1</em>) are required for acyl-CoA-dependent synthesis of ceramides containing very long acyl chain (e.g. C26) fatty acids in yeast, and a homolog of LAG<em>1</em>, ASC<em>1</em>, confers resistance in plants to fumonisin B(<em>1</em>), an inhibitor of ceramide synthesis. To understand further the mechanism of regulation of ceramide synthesis, we now characterize a mammalian homolog of LAG<em>1</em>, upstream of <em>growth</em> and <em>differentiation</em> <em>factor</em>-<em>1</em> (uog<em>1</em>). cDNA clones of uog<em>1</em> were obtained from expression sequence-tagged clones and sub-cloned into a mammalian expression vector. Transient transfection of human <em>embryonic</em> kidney 293T cells with uog<em>1</em> followed by metabolic labeling with [4,5-(3)H]sphinganine or L-3-[(3)H]serine demonstrated that uog<em>1</em> conferred fumonisin B(<em>1</em>) resistance with respect to the ability of the cells to continue to produce ceramide. Surprisingly, this ceramide was channeled into neutral glycosphingolipids but not into gangliosides. Electrospray tandem mass spectrometry confirmed the elevation in sphingolipids and revealed that the ceramides and neutral glycosphingolipids of uog<em>1</em>-transfected cells contain primarily stearic acid (C<em>1</em>8), that this enrichment was further increased by FB(<em>1</em>), and that the amount of stearic acid in sphingomyelin was also increased. UOG<em>1</em> was localized to the endoplasmic reticulum, demonstrating that the fatty acid selectivity and the fumonisin B(<em>1</em>) resistance are not due to a subcellular localization different from that found previously for ceramide synthase activity. Furthermore, in vitro assays of uog<em>1</em>-transfected cells demonstrated elevated ceramide synthase activity when stearoyl-CoA but not palmitoyl-CoA was used as substrate. We propose a role for UOG<em>1</em> in regulating C<em>1</em>8-ceramide (N-stearoyl-sphinganine) synthesis, and we note that not only is this the first case of ceramide formation in mammalian cells with such a high degree of fatty acid specificity, but also that the N-stearoyl-sphinganine produced by UOG<em>1</em> most significantly impacts neutral glycosphingolipid synthesis.