The <em>embryonic</em> programme 'epithelial-mesenchymal transition' (EMT) is thought to promote malignant tumour progression. The transcriptional repressor zinc-finger E-box binding homeobox <em>1</em> (ZEB<em>1</em>) is a crucial inducer of EMT in various human tumours, and was recently shown to promote invasion and metastasis of tumour cells. Here, we report that ZEB<em>1</em> directly suppresses transcription of microRNA-200 family members miR-<em>1</em>4<em>1</em> and miR-200c, which strongly activate epithelial <em>differentiation</em> in pancreatic, colorectal and breast cancer cells. Notably, the EMT activators transforming <em>growth</em> <em>factor</em> beta2 and ZEB<em>1</em> are the predominant targets downregulated by these microRNAs. These results indicate that ZEB<em>1</em> triggers an microRNA-mediated feedforward loop that stabilizes EMT and promotes invasion of cancer cells. Alternatively, depending on the environmental trigger, this loop might switch and induce epithelial <em>differentiation</em>, and thus explain the strong intratumorous heterogeneity observed in many human cancers.

Newborn mice homozygous for a targeted disruption of insulin-like <em>growth</em> <em>factor</em> gene (Igf-<em>1</em>) exhibit a <em>growth</em> deficiency similar in severity to that previously observed in viable Igf-2 null mutants (60% of normal birthweight). Depending on genetic background, some of the Igf-<em>1</em>(-/-) dwarfs die shortly after birth, while others survive and reach adulthood. In contrast, null mutants for the Igf<em>1</em>r gene die invariably at birth of respiratory failure and exhibit a more severe <em>growth</em> deficiency (45% normal size). In addition to generalized organ hypoplasia in Igf<em>1</em>r(-/-) embryos, including the muscles, and developmental delays in ossification, deviations from normalcy were observed in the central nervous system and epidermis. Igf-<em>1</em>(-/-)/Igf<em>1</em>r(-/-) double mutants did not differ in phenotype from Igf<em>1</em>r(-/-) single mutants, while in Igf-2(-)/Igf<em>1</em>r(-/-) and Igf-<em>1</em>(-/-)/Igf-2(-) double mutants, which are phenotypically identical, the dwarfism was further exacerbated (30% normal size). The roles of the IGFs in mouse <em>embryonic</em> development, as revealed from the phenotypic <em>differences</em> between these mutants, are discussed.

Bone morphogenetic proteins (BMPs) are multi-functional <em>growth</em> <em>factors</em> that belong to the transforming <em>growth</em> <em>factor</em> beta (TGFbeta) superfamily. The roles of BMPs in <em>embryonic</em> development and cellular functions in postnatal and adult animals have been extensively studied in recent years. Signal transduction studies have revealed that Smad<em>1</em>, 5 and 8 are the immediate downstream molecules of BMP receptors and play a central role in BMP signal transduction. Studies from transgenic and knockout mice and from animals and humans with naturally occurring mutations in BMPs and related genes have shown that BMP signaling plays critical roles in heart, neural and cartilage development. BMPs also play an important role in postnatal bone formation. BMP activities are regulated at different molecular levels. Preclinical and clinical studies have shown that BMP-2 can be utilized in various therapeutic interventions such as bone defects, non-union fractures, spinal fusion, osteoporosis and root canal surgery. Tissue-specific knockout of a specific BMP ligand, a subtype of BMP receptors or a specific signaling molecule is required to further determine the specific role of a BMP ligand, receptor or signaling molecule in a particular tissue. BMPs are members of the TGFbeta superfamily. The activity of BMPs was first identified in the <em>1</em>960s (Urist, M.R. (<em>1</em>965) "Bone formation by autoinduction", Science <em>1</em>50, 893-899), but the proteins responsible for bone induction remained unknown until the purification and sequence of bovine BMP-3 (osteogenin) and cloning of human BMP-2 and 4 in the late <em>1</em>980s (Wozney, J.M. et al. (<em>1</em>988) "Novel regulators of bone formation: molecular clones and activities", Science 242, <em>1</em>528-<em>1</em>534; Luyten, F.P. et al. (<em>1</em>989) "Purification and partial amino acid sequence of osteogenin, a protein initiating bone <em>differentiation</em>", J. Biol. Chem. 264, <em>1</em>3377-<em>1</em>3380; Wozney, J.M. (<em>1</em>992) "The bone morphogenetic protein family and osteogenesis", Mol. Reprod. Dev. 32, <em>1</em>60-<em>1</em>67). To date, around 20 BMP family members have been identified and characterized. BMPs signal through serine/threonine kinase receptors, composed of type I and II subtypes. Three type I receptors have been shown to bind BMP ligands, type IA and IB BMP receptors (BMPR-IA or ALK-3 and BMPR-IB or ALK-6) and type IA activin receptor (ActR-IA or ALK-2) (Koenig, B.B. et al. (<em>1</em>994) "Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells", Mol. Cell. Biol. <em>1</em>4, 596<em>1</em>-5974; ten Dijke, P. et al. (<em>1</em>994) "Identification of type I receptors for osteogenic protein-<em>1</em> and bone morphogenetic protein-4", J. Biol. Chem. 269, <em>1</em>6985-<em>1</em>6988; Macias-Silva, M. et al. (<em>1</em>998) "Specific activation of Smad<em>1</em> signaling pathways by the BMP7 type I receptor, ALK2", J. Biol. Chem. 273, 25628-25636). Three type II receptors for BMPs have also been identified and they are type II BMP receptor (BMPR-II) and type II and IIB activin receptors (ActR-II and ActR-IIB) (Yamashita, H. et al. (<em>1</em>995) "Osteogenic protein-<em>1</em> binds to activin type II receptors and induces certain activin-like effects", J. Cell. Biol. <em>1</em>30, 2<em>1</em>7-226; Rosenzweig, B.L. et al. (<em>1</em>995) "Cloning and characterization of a human type II receptor for bone morphogenetic proteins", Proc. Natl Acad. Sci. USA 92, 7632-7636; Kawabata, M. et al. (<em>1</em>995) "Cloning of a novel type II serine/threonine kinase receptor through interaction with the type I transforming <em>growth</em> <em>factor</em>-beta receptor", J. Biol. Chem. 270, 5625-5630). Whereas BMPR-IA, IB and II are specific to BMPs, ActR-IA, II and IIB are also signaling receptors for activins. These receptors are expressed differentially in various tissues. Type I and II BMP receptors are both indispensable for signal transduction. After ligand binding they form a heterotetrameric-activated receptor complex consisting of two pairs of a type I and II receptor complex (Moustakas, A. and C.H. Heldi (2002) "From mono- to oligo-Smads: the heart of the matter in TGFbeta signal transduction" Genes Dev. <em>1</em>6, 67-87<em>1</em>). The type I BMP receptor substrates include a protein family, the Smad proteins, that play a central role in relaying the BMP signal from the receptor to target genes in the nucleus. Smad<em>1</em>, 5 and 8 are phosphorylated by BMP receptors in a ligand-dependent manner (Hoodless, P.A. et al. (<em>1</em>996) "MADR<em>1</em>, a MAD-related protein that functions in BMP2 signaling pathways", Cell 85, 489-500; Chen Y. et al. (<em>1</em>997) "Smad8 mediates the signaling of the receptor serine kinase", Proc. Natl Acad. Sci. USA 94, <em>1</em>2938-<em>1</em>2943; Nishimura R. et al. (<em>1</em>998) "Smad5 and DPC4 are key molecules in mediating BMP-2-induced osteoblastic <em>differentiation</em> of the pluripotent mesenchymal precursor cell line C2C<em>1</em>2", J. Biol. Chem. 273, <em>1</em>872-<em>1</em>879). After release from the receptor, the phosphorylated Smad proteins associate with the related protein Smad4, which acts as a shared partner. This complex translocates into the nucleus and participates in gene transcription with other transcription <em>factors</em> (). A significant advancement about the understanding of in vivo functions of BMP ligands, receptors and signaling molecules has been achieved in recent years.

The functional heart is comprised of distinct mesoderm-derived lineages including cardiomyocytes, endothelial cells and vascular smooth muscle cells. Studies in the mouse embryo and the mouse <em>embryonic</em> stem cell <em>differentiation</em> model have provided evidence indicating that these three lineages develop from a common Flk-<em>1</em>(+) (kinase insert domain protein receptor, also known as Kdr) cardiovascular progenitor that represents one of the earliest stages in mesoderm specification to the cardiovascular lineages. To determine whether a comparable progenitor is present during human cardiogenesis, we analysed the development of the cardiovascular lineages in human <em>embryonic</em> stem cell <em>differentiation</em> cultures. Here we show that after induction with combinations of activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast <em>growth</em> <em>factor</em> (bFGF, also known as FGF2), vascular endothelial <em>growth</em> <em>factor</em> (VEGF, also known as VEGFA) and dickkopf homolog <em>1</em> (DKK<em>1</em>) in serum-free media, human <em>embryonic</em>-stem-cell-derived embryoid bodies generate a KDR(low)/C-KIT(CD<em>1</em><em>1</em>7)(neg) population that displays cardiac, endothelial and vascular smooth muscle potential in vitro and, after transplantation, in vivo. When plated in monolayer cultures, these KDR(low)/C-KIT(neg) cells differentiate to generate populations consisting of greater than 50% contracting cardiomyocytes. Populations derived from the KDR(low)/C-KIT(neg) fraction give rise to colonies that contain all three lineages when plated in methylcellulose cultures. Results from limiting dilution studies and cell-mixing experiments support the interpretation that these colonies are clones, indicating that they develop from a cardiovascular colony-forming cell. Together, these findings identify a human cardiovascular progenitor that defines one of the earliest stages of human cardiac development.

The vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and its high-affinity binding receptors, the tyrosine kinases Flt-<em>1</em> and Flk-<em>1</em>, are thought to be important for the development of <em>embryonic</em> vasculature. Here we report that Flt-<em>1</em> is essential for the organization of <em>embryonic</em> vasculature, but is not essential for endothelial cell <em>differentiation</em>. Mouse embryos homozygous for a targeted mutation in the flt-<em>1</em> locus, flt-<em>1</em>lcz, formed endothelial cells in both <em>embryonic</em> and extra-<em>embryonic</em> regions, but assembled these cells into abnormal vascular channels and died in utero at mid-somite stages. At earlier stages, the blood islands of flt-<em>1</em>lcz homozygotes were abnormal, with angioblasts in the interior as well as on the periphery. We suggest that the Flt-<em>1</em> signalling pathway may regulate normal endothelial cell-cell or cell-matrix interactions during vascular development.

The transcriptional response to lowered oxygen levels is mediated by the hypoxia-inducible transcription <em>factor</em> (HIF-<em>1</em>), a heterodimer consisting of the constitutively expressed aryl hydrocarbon receptor nuclear translocator (ARNT) and the hypoxic response <em>factor</em> HIF-<em>1</em>alpha. To study the role of the transcriptional hypoxic response in vivo we have targeted the murine HIF-<em>1</em>alpha gene. Loss of HIF-<em>1</em>alpha in <em>embryonic</em> stem (ES) cells dramatically retards solid tumor <em>growth</em>; this is correlated with a reduced capacity to release the angiogenic <em>factor</em> vascular endothelial <em>growth</em> <em>factor</em> (VEGF) during hypoxia. HIF-<em>1</em>alpha null mutant embryos exhibit clear morphological <em>differences</em> by <em>embryonic</em> day (E) 8.0, and by E8.5 there is a complete lack of cephalic vascularization, a reduction in the number of somites, abnormal neural fold formation and a greatly increased degree of hypoxia (measured by the nitroimidazole EF5). These data demonstrate the essential role of HIF-<em>1</em>alpha in controlling both <em>embryonic</em> and tumorigenic responses to variations in microenvironmental oxygenation.

Accumulating evidence implicates the transcription <em>factor</em> NF-kappaB as a positive mediator of cell <em>growth</em>, but the molecular mechanism(s) involved in this process remains largely unknown. Here we use both a skeletal muscle <em>differentiation</em> model and normal diploid fibroblasts to gain insight into how NF-kappaB regulates cell <em>growth</em> and <em>differentiation</em>. Results obtained with the C2C<em>1</em>2 myoblast cell line demonstrate that NF-kappaB functions as an inhibitor of myogenic <em>differentiation</em>. Myoblasts generated to lack NF-kappaB activity displayed defects in cellular proliferation and cell cycle exit upon <em>differentiation</em>. An analysis of cell cycle markers revealed that NF-kappaB activates cyclin D<em>1</em> expression, and the results showed that this regulatory pathway is one mechanism by which NF-kappaB inhibits myogenesis. NF-kappaB regulation of cyclin D<em>1</em> occurs at the transcriptional level and is mediated by direct binding of NF-kappaB to multiple sites in the cyclin D<em>1</em> promoter. Using diploid fibroblasts, we demonstrate that NF-kappaB is required to induce cyclin D<em>1</em> expression and pRb hyperphosphorylation and promote G(<em>1</em>)-to-S progression. Consistent with results obtained with the C2C<em>1</em>2 <em>differentiation</em> model, we show that NF-kappaB also promotes cell <em>growth</em> in <em>embryonic</em> fibroblasts, correlating with its regulation of cyclin D<em>1</em>. These data therefore identify cyclin D<em>1</em> as an important transcriptional target of NF-kappaB and reveal a mechanism to explain how NF-kappaB is involved in the early phases of the cell cycle to regulate cell <em>growth</em> and <em>differentiation</em>.

Fracture healing is a specialized post-natal repair process that recapitulates aspects of embryological skeletal development. While many of the molecular mechanisms that control cellular <em>differentiation</em> and <em>growth</em> during embryogenesis recur during fracture healing, these processes take place in a post-natal environment that is unique and distinct from those which exist during embryogenesis. This Prospect Article will highlight a number of central biological processes that are believed to be crucial in the <em>embryonic</em> <em>differentiation</em> and <em>growth</em> of skeletal tissues and review the functional role of these processes during fracture healing. Specific aspects of fracture healing that will be considered in relation to embryological development are: (<em>1</em>) the anatomic structure of the fracture callus as it evolves during healing; (2) the origins of stem cells and morphogenetic signals that facilitate the repair process; (3) the role of the biomechanical environment in controlling cellular <em>differentiation</em> during repair; (4) the role of three key groups of soluble <em>factors</em>, pro-inflammatory cytokines, the TGF-beta superfamily, and angiogenic <em>factors</em>, during repair; and (5) the relationship of the genetic components that control bone mass and remodeling to the mechanisms that control skeletal tissue repair in response to fracture.

Pluripotent <em>embryonic</em> stem (ES) cells must select between alternative fates of self-replication and lineage commitment during continuous proliferation. Here, we delineate the role of autocrine production of fibroblast <em>growth</em> <em>factor</em> 4 (Fgf4) and associated activation of the Erk<em>1</em>/2 (Mapk3/<em>1</em>) signalling cascade. Fgf4 is the major stimulus activating Erk in mouse ES cells. Interference with FGF or Erk activity using chemical inhibitors or genetic ablations does not impede propagation of undifferentiated ES cells. Instead, such manipulations restrict the ability of ES cells to commit to <em>differentiation</em>. ES cells lacking Fgf4 or treated with FGF receptor inhibitors resist neural and mesodermal induction, and are refractory to BMP-induced non-neural <em>differentiation</em>. Lineage commitment potential of Fgf4-null cells is restored by provision of FGF protein. Thus, FGF enables rather than antagonises the <em>differentiation</em> activity of BMP. The key downstream role of Erk signalling is revealed by examination of Erk2-null ES cells, which fail to undergo either neural or mesodermal <em>differentiation</em> in adherent culture, and retain expression of pluripotency markers Oct4, Nanog and Rex<em>1</em>. These findings establish that Fgf4 stimulation of Erk<em>1</em>/2 is an autoinductive stimulus for naïve ES cells to exit the self-renewal programme. We propose that the Erk cascade directs transition to a state that is responsive to inductive cues for germ layer segregation. Consideration of Erk signalling as a primary trigger that potentiates lineage commitment provides a context for reconciling disparate views on the contribution of FGF and BMP pathways during germ layer specification in vertebrate embryos.

Cell-tracing studies in the mouse indicate that the cardiac lineage arises from a population that expresses the vascular endothelial <em>growth</em> <em>factor</em> receptor 2 (VEGFR2, Flk-<em>1</em>), suggesting that it may develop from a progenitor with vascular potential. Using the <em>embryonic</em> stem (ES) cell <em>differentiation</em> model, we have identified a cardiovascular progenitor based on the temporal expression of the primitive streak (PS) marker brachyury and Flk-<em>1</em>. Comparable progenitors could also be isolated from head-fold stage embryos. When cultured with cytokines known to function during cardiogenesis, individual cardiovascular progenitors generated colonies that displayed cardiomyocyte, endothelial, and vascular smooth muscle (VSM) potential. Isolation and characterization of this previously unidentified population suggests that the mammalian cardiovascular system develops from multipotential progenitors.

Sphingosine-<em>1</em>-phosphate (S<em>1</em>P), an important sphingolipid metabolite, regulates diverse cellular processes, including cell survival, <em>growth</em>, and <em>differentiation</em>. Here we show that S<em>1</em>P signaling is critical for neural and vascular development. Sphingosine kinase-null mice exhibited a deficiency of S<em>1</em>P which severely disturbed neurogenesis, including neural tube closure, and angiogenesis and caused <em>embryonic</em> lethality. A dramatic increase in apoptosis and a decrease in mitosis were seen in the developing nervous system. S<em>1</em>P(<em>1</em>) receptor-null mice also showed severe defects in neurogenesis, indicating that the mechanism by which S<em>1</em>P promotes neurogenesis is, in part, signaling from the S<em>1</em>P(<em>1</em>) receptor. Thus, S<em>1</em>P joins a <em>growing</em> list of signaling molecules, such as vascular endothelial <em>growth</em> <em>factor</em>, which regulate the functionally intertwined pathways of angiogenesis and neurogenesis. Our findings also suggest that exploitation of this potent neuronal survival pathway could lead to the development of novel therapeutic approaches for neurological diseases.

daf-<em>1</em>6 is a forkhead-type transcription <em>factor</em>, functioning downstream of insulin-like signals, and is known to be critical to the regulation of life span in Caenorhabditis elegans. Mammalian DAF-<em>1</em>6 homologues include AFX, FKHR and FKHRL<em>1</em>, which contain a conserved forkhead domain and three putative phosphorylation sites for the Ser/Thr kinase Akt/protein kinase B (PKB), as well as for DAF-<em>1</em>6. To assess the function of the homologues, we examined tissue distribution patterns of mRNAs for DAF-<em>1</em>6 homologues in mice. In the embryos, expressions of AFX, FKHR and FKHRL<em>1</em> mRNAs were complementary to each other and were highest in muscle, adipose tissue and <em>embryonic</em> liver. The characteristic expression pattern remained in the adult, except that signals of FKHRL<em>1</em> became evident in more tissues, including the brain. In order to clarify whether each DAF-<em>1</em>6 homologue had different target genes, we determined the consensus sequences for the binding of DAF-<em>1</em>6 and the mouse homologues. The binding sequences for all four proteins shared a core sequence, TTGTTTAC, daf-<em>1</em>6 family protein-binding element (DBE) binding protein. However, electrophoretic mobility shift assay showed that the binding affinity of DAF-<em>1</em>6 homologues to the core sequence was stronger than that to the insulin-responsive element in the insulin-like <em>growth</em> <em>factor</em> binding protein-<em>1</em> promoter region, which has been identified as a binding sequence for them. We identified one copy of the DBE upstream of the first exon of sod-3 by searching the genomic database of C. elegans. Taken together, DAF-<em>1</em>6 homologues can fundamentally regulate the common target genes in insulin-responsive tissues and the specificity to target genes of each protein is partially determined by the <em>differences</em> in their expression patterns.

Insulin-like <em>growth</em> <em>factor</em> II (IGF-II) is a peptide <em>growth</em> <em>factor</em> that is homologous to both insulin-like <em>growth</em> <em>factor</em> I (IGF-I) and insulin and plays an important role in <em>embryonic</em> development and carcinogenesis. IGF-II is believed to mediate its cellular signaling via the transmembrane tyrosine kinase type <em>1</em> insulin-like <em>growth</em> <em>factor</em> receptor (IGF-I-R), which is also the receptor for IGF-I. Earlier studies with both cultured cells and transgenic mice, however, have suggested that in the embryo the insulin receptor (IR) may also be a receptor for IGF-II. In most cells and tissues, IR binds IGF-II with relatively low affinity. The IR is expressed in two isoforms (IR-A and IR-B) differing by <em>1</em>2 amino acids due to the alternative splicing of exon <em>1</em><em>1</em>. In the present study we found that IR-A but not IR-B bound IGF-II with an affinity close to that of insulin. Moreover, IGF-II bound to IR-A with an affinity equal to that of IGF-II binding to the IGF-I-R. Activation of IR-A by insulin led primarily to metabolic effects, whereas activation of IR-A by IGF-II led primarily to mitogenic effects. These <em>differences</em> in the biological effects of IR-A when activated by either IGF-II or insulin were associated with differential recruitment and activation of intracellular substrates. IR-A was preferentially expressed in fetal cells such as fetal fibroblasts, muscle, liver and kidney and had a relatively increased proportion of isoform A. IR-A expression was also increased in several tumors including those of the breast and colon. These data indicate, therefore, that there are two receptors for IGF-II, both IGF-I-R and IR-A. Further, they suggest that interaction of IGF-II with IR-A may play a role both in fetal <em>growth</em> and cancer biology.

We report that <em>embryonic</em> stem cells efficiently undergo <em>differentiation</em> in vitro to mesoderm and hematopoietic cells and that this in vitro system recapitulates days 6.5 to 7.5 of mouse hematopoietic development. <em>Embryonic</em> stem cells differentiated as embryoid bodies (EBs) develop erythroid precursors by day 4 of <em>differentiation</em>, and by day 6, more than 85% of EBs contain such cells. A comparative reverse transcriptase-mediated polymerase chain reaction profile of marker genes for primitive endoderm (collagen alpha IV) and mesoderm (Brachyury) indicates that both cell types are present in the developing EBs as well in normal embryos prior to the onset of hematopoiesis. GATA-<em>1</em>, GATA-3, and vav are expressed in both the EBs and embryos just prior to and/or during the early onset of hematopoiesis, indicating that they could play a role in the early stages of hematopoietic development both in vivo and in vitro. The initial stages of hematopoietic development within the EBs occur in the absence of added <em>growth</em> <em>factors</em> and are not significantly influenced by the addition of a broad spectrum of <em>factors</em>, including interleukin-3 (IL-3), IL-<em>1</em>, IL-6, IL-<em>1</em><em>1</em>, erythropoietin, and Kit ligand. At days <em>1</em>0 and <em>1</em>4 of <em>differentiation</em>, EB hematopoiesis is significantly enhanced by the addition of both Kit ligand and IL-<em>1</em><em>1</em> to the cultures. Kinetic analysis indicates that hematopoietic precursors develop within the EBs in an ordered pattern. Precursors of the primitive erythroid lineage appear first, approximately 24 h before precursors of the macrophage and definitive erythroid lineages. Bipotential neutrophil/macrophage and multilineage precursors appear next, and precursors of the mast cell lineage develop last. The kinetics of precursor development, as well as the <em>growth</em> <em>factor</em> responsiveness of these early cells, is similar to that found in the yolk sac and early fetal liver, indicating that the onset of hematopoiesis within the EBs parallels that found in the embryo.

The TGF-beta superfamily of proteins regulates many different biological processes, including cell <em>growth</em>, <em>differentiation</em> and <em>embryonic</em> pattern formation. TGF-beta-like <em>factors</em> signal across cell membranes through complexes of transmembrane receptors known as type I and type II serine/threonine-kinase receptors, which in turn activate the SMAD signalling pathway. On the inside of the cell membrane, a receptor-regulated class of SMADs are phosphorylated by the type-I-receptor kinase. In this way, receptors for different <em>factors</em> are able to pass on specific signals along the pathway: for example, receptors for bone morphogenetic protein (BMP) target SMADs <em>1</em>, 5 and 8, whereas receptors for activin and TGF-beta target SMADs 2 and 3. Phosphorylation of receptor-regulated SMADs induces their association with Smad4, the 'common-partner' SMAD, and stimulates accumulation of this complex in the nucleus, where it regulates transcriptional responses. Here we describe Smurf<em>1</em>, a new member of the Hect family of E3 ubiquitin ligases. Smurf<em>1</em> selectively interacts with receptor-regulated SMADs specific for the BMP pathway in order to trigger their ubiquitination and degradation, and hence their inactivation. In the amphibian Xenopus laevis, Smurf<em>1</em> messenger RNA is localized to the animal pole of the egg; in Xenopus embryos, ectopic Smurf<em>1</em> inhibits the transmission of BMP signals and thereby affects pattern formation. Smurf<em>1</em> also enhances cellular responsiveness to the Smad2 (activin/TGF-beta) pathway. Thus, targeted ubiquitination of SMADs may serve to control both <em>embryonic</em> development and a wide variety of cellular responses to TGF-beta signals.

Smooth muscle cells switch between differentiated and proliferative phenotypes in response to extracellular cues, but the transcriptional mechanisms that confer such phenotypic plasticity remain unclear. Serum response <em>factor</em> (SRF) activates genes involved in smooth muscle <em>differentiation</em> and proliferation by recruiting muscle-restricted co<em>factors</em>, such as the transcriptional coactivator myocardin, and ternary complex <em>factors</em> (TCFs) of the ETS-domain family, respectively. Here we show that <em>growth</em> signals repress smooth muscle genes by triggering the displacement of myocardin from SRF by Elk-<em>1</em>, a TCF that acts as a myogenic repressor. The opposing influences of myocardin and Elk-<em>1</em> on smooth muscle gene expression are mediated by structurally related SRF-binding motifs that compete for a common docking site on SRF. A mutant smooth muscle promoter, retaining responsiveness to myocardin and SRF but defective in TCF binding, directs ectopic transcription in the <em>embryonic</em> heart, demonstrating a role for TCFs in suppression of smooth muscle gene expression in vivo. We conclude that <em>growth</em> and developmental signals modulate smooth muscle gene expression by regulating the association of SRF with antagonistic co<em>factors</em>.

Distinctive properties of stem cells are not autonomously achieved, and recent evidence points to a level of external control from the microenvironment. Here, we demonstrate that self-renewal and pluripotent properties of human <em>embryonic</em> stem (ES) cells depend on a dynamic interplay between human ES cells and autologously derived human ES cell fibroblast-like cells (hdFs). Human ES cells and hdFs are uniquely defined by insulin-like <em>growth</em> <em>factor</em> (IGF)- and fibroblast <em>growth</em> <em>factor</em> (FGF)-dependence. IGF <em>1</em> receptor (IGF<em>1</em>R) expression was exclusive to the human ES cells, whereas FGF receptor <em>1</em> (FGFR<em>1</em>) expression was restricted to surrounding hdFs. Blocking the IGF-II/IGF<em>1</em>R pathway reduced survival and clonogenicity of human ES cells, whereas inhibition of the FGF pathway indirectly caused <em>differentiation</em>. IGF-II is expressed by hdFs in response to FGF, and alone was sufficient in maintaining human ES cell cultures. Our study demonstrates a direct role of the IGF-II/IGF<em>1</em>R axis on human ES cell physiology and establishes that hdFs produced by human ES cells themselves define the stem cell niche of pluripotent human stem cells.

Human <em>embryonic</em> stem (ES) cells are undifferentiated, pluripotent cells that can be maintained indefinitely in culture. Here we demonstrate that human ES cells differentiate to hematopoietic precursor cells when cocultured with the murine bone marrow cell line S<em>1</em>7 or the yolk sac endothelial cell line C<em>1</em>66. This hematopoietic <em>differentiation</em> requires fetal bovine serum, but no other exogenous cytokines. ES cell-derived hematopoietic precursor cells express the cell surface antigen CD34 and the hematopoietic transcription <em>factors</em> TAL-<em>1</em>, LMO-2, and GATA-2. When cultured on semisolid media with hematopoietic <em>growth</em> <em>factors</em>, these hematopoietic precursor cells form characteristic myeloid, erythroid, and megakaryocyte colonies. Selection for CD34(+) cells derived from human ES cells enriches for hematopoietic colony-forming cells, similar to CD34 selection of primary hematopoietic tissue (bone marrow, umbilical cord blood). More terminally differentiated hematopoietic cells derived from human ES cells under these conditions also express normal surface antigens: glycophorin A on erythroid cells, CD<em>1</em>5 on myeloid cells, and CD4<em>1</em> on megakaryocytes. The in vitro <em>differentiation</em> of human ES cells provides an opportunity to better understand human hematopoiesis and could lead to a novel source of cells for transfusion and transplantation therapies.

Bone morphogenetic proteins (BMPs) are secreted proteins that interact with cell-surface receptors and are believed to play a variety of important roles during vertebrate embryogenesis. Bmpr, also known as ALK-3 and Brk-<em>1</em>, encodes a type I transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) family receptor for BMP-2 and BMP-4. Bmpr is expressed ubiquitously during early mouse embryogenesis and in most adult mouse tissues. To study the function of Bmpr during mammalian development, we generated Bmpr-mutant mice. After <em>embryonic</em> day 9.5 (E9.5), no homozygous mutants were recovered from heterozygote matings. Homozygous mutants with morphological defects were first detected at E7.0 and were smaller than normal. Morphological and molecular examination demonstrated that no mesoderm had formed in the mutant embryos. The <em>growth</em> characteristics of homozygous mutant blastocysts cultured in vitro were indistinguishable from those of controls; however, <em>embryonic</em> ectoderm (epiblast) cell proliferation was reduced in all homozygous mutants at E6.5 before morphological abnormalities had become prominent. Teratomas arising from E7.0 mutant embryos contained derivatives from all three germ layers but were smaller and gave rise to fewer mesodermal cell types, such as muscle and cartilage, than controls. These results suggest that signaling through this type I BMP-2/4 receptor is not necessary for preimplantation or for initial postimplantation development but may be essential for the inductive events that lead to the formation of mesoderm during gastrulation and later for the <em>differentiation</em> of a subset of mesodermal cell types.

The <em>differentiation</em> capacity of human <em>embryonic</em> stem cells (hESCs) holds great promise for therapeutic applications. We report a novel three-stage method to efficiently direct the <em>differentiation</em> of human <em>embryonic</em> stem cells into hepatic cells in serum-free medium. Human ESCs were first differentiated into definitive endoderm cells by 3 days of Activin A treatment. Next, the presence of fibroblast <em>growth</em> <em>factor</em>-4 and bone morphogenetic protein-2 in the culture medium for 5 days induced efficient hepatic <em>differentiation</em> from definitive endoderm cells. Approximately 70% of the cells expressed the hepatic marker albumin. After <em>1</em>0 days of further in vitro maturation, these cells expressed the adult liver cell markers tyrosine aminotransferase, tryptophan oxygenase 2, phosphoenolpyruvate carboxykinase (PEPCK), Cyp7A<em>1</em>, Cyp3A4 and Cyp2B6. Furthermore, these cells exhibited functions associated with mature hepatocytes including albumin secretion, glycogen storage, indocyanine green, and low-density lipoprotein uptake, and inducible cytochrome P450 activity. When transplanted into CCl4 injured severe combined immunodeficiency mice, these cells integrated into the mouse liver and expressed human alpha-<em>1</em> antitrypsin for at least 2 months. In addition, we found that the hESC-derived hepatic cells were readily infected by human immunodeficiency virus-hepatitis C virus pseudotype viruses.

CONCLUSIONS

We have developed an efficient way to direct the differentiation of human embryonic stem cells into cells that exhibit characteristics of mature hepatocytes. Our studies should facilitate searching the molecular mechanisms underlying human liver development, and form the basis for hepatocyte transplantation and drug tests.

We have explored the role of fibroblast <em>growth</em> <em>factor</em> receptor <em>1</em> (FGFR-<em>1</em>) in early <em>embryonic</em> development using three experimental systems: genetically deficient mice, in vitro blastocyst culture, and FGFR-<em>1</em>-deficient <em>embryonic</em> stem cells. Using these systems, we demonstrate that FGFR-<em>1</em> is required for proper <em>embryonic</em> cell proliferation and for the correct axial organization of early postimplantation embryos but not for mesoderm formation. FGFR-<em>1</em>-deficient embryos display severe <em>growth</em> retardation both in vitro and in vivo and die prior to or during gastrulation. Although these mutants can form nonaxial tissues, such as the allantois, amnion, and yolk sac mesoderm, they display defective patterning of the primitive streak and other axial structures, and frequently exhibit truncations or disorganization of posterior <em>embryonic</em> regions. Such abnormalities are unlikely to be caused by intrinsic blocks in mesodermal <em>differentiation</em>, as FGFR-<em>1</em>-deficient ES cell lines form teratomas consisting of many mesodermal cell types.

The proto-oncogene c-fos is the cellular homologue of v-fos originally isolated from murine osteosarcoma. Fos protein is a major component of the AP-<em>1</em> transcription <em>factor</em> complex, which includes members of the jun family. Stable expression of c-fos in mice has been demonstrated in developing bones and teeth, haematopoietic cells, germ cells and in the central nervous system. It has been proposed that c-fos has an important role in signal transduction, cell proliferation and <em>differentiation</em>. We have previously demonstrated that overexpression of c-fos in transgenic and chimaeric mice specifically affects bone, cartilage and haematopoietic cell development. To understand better the function of c-fos in vivo, we used gene targeting in <em>embryonic</em> stem cells to generate cells and mice lacking c-fos. Here we report that heterozygous fos +/- mice appear normal, although females exhibit a distorted transmission frequency. All homozygous fos -/- mice are <em>growth</em>-retarded, develop osteopetrosis with deficiencies in bone remodelling and tooth eruption, and have altered haematopoiesis. These data define the c-Fos protein as an essential molecule for the development of specific cellular compartments.

We report here the isolation of a population of non-transformed pluripotent human cells from bone marrow after a unique expansion/selection procedure. This procedure was designed to provide conditions resembling the in vivo microenvironment that is home for the most-primitive stem cells. Marrow-adherent and -nonadherent cells were co-cultured on fibronectin, at low oxygen tension, for <em>1</em>4 days. Colonies of small adherent cells were isolated and further expanded on fibronectin at low density, low oxygen tension with 2% fetal bovine serum. They expressed high levels of CD29, CD63, CD8<em>1</em>, CD<em>1</em>22, CD<em>1</em>64, hepatocyte <em>growth</em> <em>factor</em> receptor (cMet), bone morphogenetic protein receptor <em>1</em>B (BMPR<em>1</em>B), and neurotrophic tyrosine kinase receptor 3 (NTRK3) and were negative for CD34, CD36, CD45, CD<em>1</em><em>1</em>7 (cKit) and HLADR. The <em>embryonic</em> stem cell markers Oct-4 and Rex-<em>1</em>, and telomerase were expressed in all cultures examined. Cell-doubling time was 36 to 72 hours, and cells have been expanded in culture for more than 50 population doublings. This population of cells was consistently isolated from men and women of ages ranging from 3- to 72-years old. Colonies of cells expressed numerous markers found among <em>embryonic</em> stem cells as well as mesodermal-, endodermal- and ectodermal-derived lineages. They have been differentiated to bone-forming osteoblasts, cartilage-forming chondrocytes, fat-forming adipocytes and neural cells and to attachment-independent spherical clusters expressing genes associated with pancreatic islets. Based on their unique characteristics and properties, we refer to them as human marrow-isolated adult multilineage inducible cells, or MIAMI cells. MIAMI cells proliferate extensively without evidence of senescence or loss of <em>differentiation</em> potential and thus may represent an ideal candidate for cellular therapies of inherited or degenerative diseases.

The various cell types in a multicellular animal differentiate on a predictable schedule but the mechanisms responsible for timing cell <em>differentiation</em> are largely unknown. We have studied a population of bipotential glial (O-2A) progenitor cells in the developing rat optic nerve that gives rise to oligodendrocytes beginning at birth and to type-2 astrocytes beginning in the second postnatal week. Whereas, in vivo, these O-2A progenitor cells proliferate and give rise to postimitotic oligodendrocytes over several weeks, in serum-free (or low-serum) culture they stop dividing prematurely and differentiate into oligodendrocytes within two or three days. The normal timing of oligodendrocyte development can be restored if <em>embryonic</em> optic-nerve cells are cultured in medium conditioned by type-<em>1</em> astrocytes, the first glial cells to differentiate in the nerve: in this case the progenitor cells continue to proliferate, the first oligodendrocytes appear on the equivalent of the day of birth, and new oligodendrocytes continue to develop over several weeks, just as in vivo. Here we show that platelet-derived <em>growth</em> <em>factor</em> (PDGF) can replace type-<em>1</em>-astrocyte-conditioned medium in restoring the normal timing of oligodendrocyte <em>differentiation</em> in vitro and that anti-PDGF antibodies inhibit this property of the appropriately conditioned medium. We also show that PDGF is present in the developing optic nerve. These findings suggest that type-<em>1</em>-astrocyte-derived PDGF drives the clock that times oligodendrocyte development.