Histone methylation is an important regulator of gene expression; its coordinated activity is critical in complex developmental processes such as hematopoiesis. Disruptor of telomere silencing <em>1</em>-like (DOT<em>1</em>L) is a unique histone methyltransferase that specifically methylates histone H3 at lysine 79. We analyzed Dot<em>1</em>L-mutant mice to determine influence of this enzyme on <em>embryonic</em> hematopoiesis. Mutant mice developed more slowly than wild-type embryos and died between <em>embryonic</em> days <em>1</em>0.5 and <em>1</em>3.5, displaying a striking anemia, especially apparent in small vessels of the yolk sac. Further, a severe, selective defect in erythroid, but not myeloid, <em>differentiation</em> was observed. Erythroid progenitors failed to develop normally, showing retarded progression through the cell cycle, accumulation during G₀/G₁ stage, and marked increase in apoptosis in response to erythroid <em>growth</em> <em>factors</em>. GATA2, a <em>factor</em> essential for early erythropoiesis, was significantly reduced in Dot<em>1</em>L-deficient cells, whereas expression of PU.<em>1</em>, a transcription <em>factor</em> that inhibits erythropoiesis and promotes myelopoiesis, was increased. These data suggest a model whereby DOT<em>1</em>L-dependent lysine 79 of histone H3 methylation serves as a critical regulator of a <em>differentiation</em> switch during early hematopoiesis, regulating steady-state levels of GATA2 and PU.<em>1</em> transcription, thus controlling numbers of circulating erythroid and myeloid cells.

Dissection of cardiomyocyte <em>differentiation</em> process at the cellular level is indispensable in the research for cardiac development and regeneration. Previously, we have established an <em>embryonic</em> stem cell <em>differentiation</em> system that reproduces early vascular development from progenitor cells that express Flk<em>1</em>, a vascular endothelial <em>growth</em> <em>factor</em> receptor, by the combinatory application of 2-dimensional culture and flowcytometry. Here we show that cardiomyocytes can be successfully induced from a single Flk<em>1</em>+ cell on 2-dimensional culture, enabling the direct observation of differentiating cardiomyocytes and the prospective identification of cardiac progenitor potentials. Flk<em>1</em>+ cells could give rise to cardiomyocytes, as well as endothelial cells, from a single cell by the co-culture on OP9 stroma cells in a fusion-independent manner. Among the cell populations in intermediate stages from Flk<em>1</em>+ cells to cardiomyocytes, Flk<em>1</em>+/CXCR4+/vascular endothelial cadherin- cells were cardiac-specific progenitors at the single cell level. Noggin, a bone morphogenetic protein inhibitor, abolished cardiomyocyte <em>differentiation</em> by inhibiting the cardiac progenitor induction. However, wnt inhibitors Dkk-<em>1</em> or Frizzled-8/Fc chimeric protein augmented, but wnt3a inhibited, cardiomyocyte <em>differentiation</em>. In vitro reproduction of cardiomyocyte <em>differentiation</em> process should be a potent tool for the cellular and molecular elucidation of cardiac development, which would provide various targets for cardiac regeneration.

Insulin-like <em>growth</em> <em>factor</em>-I (IGF-I), a 70-amino acid-protein structurally similar to insulin, promotes cell proliferation and <em>differentiation</em> in multiple tissues. Most of its effects are mediated by the Type I IGF receptor (IGF-IR), a heterotetramer that has tyrosine kinase activity and phosphorylates insulin receptor substrates (IRS-<em>1</em> and 2) which leads to the activation of two downstream signaling cascades: the MAP kinase and the phosphatidylinositol 3-kinase (P3K) cascades. The <em>growth</em>-promoting effects of IGF-I are prominent in the nervous system, qualifying this molecule as a neurotrophin. Although the primary regulator of IGF-I expression is <em>growth</em> hormone (GH), the developmental expression of IGF-I in various tissues precedes that of GH, supporting an independent role of IGF-I in <em>embryonic</em> and fetal life [<em>1</em>]. This review will examine the effect of IGF-I on central nervous system (CNS) development. The specialized structure of the CNS is the product of a complex series of biological events which result from the interaction between the cells' genetic program and environmental influences. CNS development begins in the embryo with dorsal ectodermal cell proliferation to form the neural plate, and, with its closure, the neural tube, followed by the rapid division of pluripotential cells, their migration to the periphery of the neural tube, and <em>differentiation</em> into neural or glial cells. During the latter stages, cells form special structures such as nuclei, ganglia, cerebral cortical layers, and they also develop a network with their cytoplasmic extensions, neurites. Many more cells and connections are generated in fetal life than are found in the mature organism. This excessive production of some cell groups and neurites may compensate for tissue loss due to various injuries, and their selective elimination also constitutes an efficient way to organize the architecture of the CNS. This elimination is believed to be accomplished by apoptosis. The cells' intrinsic program for development includes the expression of various genes at different times. Environmental influences, such as extracellular matrix (ECM) molecules that attract or repel cells, afferent inputs, and target-derived diffusible molecules modify and modulate cellular behavior. IGF-I is among the molecules which affect several steps involved in development.

Melanocytes characterized by the activities of tyrosinase, tyrosinase-related protein (TRP)-<em>1</em> and TRP-2 as well as by melanosomes and dendrites are located mainly in the epidermis, dermis and hair bulb of the mammalian skin. Melanocytes differentiate from melanoblasts, undifferentiated precursors, derived from <em>embryonic</em> neural crest cells. Because hair bulb melanocytes are derived from epidermal melanoblasts and melanocytes, the mechanism of the regulation of the proliferation and <em>differentiation</em> of epidermal melanocytes should be clarified. The regulation by the tissue environment, especially by keratinocytes is indispensable in addition to the regulation by genetic <em>factors</em> in melanocytes. Recent advances in the techniques of tissue culture and biochemistry have enabled us to clarify <em>factors</em> derived from keratinocytes. Alpha-melanocyte-stimulating hormone, adrenocorticotrophic hormone, basic fibroblast <em>growth</em> <em>factor</em>, nerve <em>growth</em> <em>factor</em>, endothelins, granulocyte-macrophage colony-stimulating <em>factor</em>, steel <em>factor</em>, leukemia inhibitory <em>factor</em> and hepatocyte <em>growth</em> <em>factor</em> have been suggested to be the keratinocyte-derived <em>factors</em> and to regulate the proliferation and/or <em>differentiation</em> of mammalian epidermal melanocytes. Numerous <em>factors</em> may be produced in and released from keratinocytes and be involved in regulating the proliferation and <em>differentiation</em> of mammalian epidermal melanocytes through receptor-mediated signaling pathways.

Nuclear reprogramming of somatic tissue enables derivation of induced pluripotent stem (iPS) cells from an autologous, non-<em>embryonic</em> origin. The purpose of this study was to establish efficient protocols for lineage specification of human iPS cells into functional glucose-responsive, insulin-producing progeny. We generated human iPS cells, which were then guided with recombinant <em>growth</em> <em>factors</em> that mimic the essential signaling for pancreatic development. Reprogrammed with four stemness <em>factors</em>, human fibroblasts were here converted into authentic iPS cells. Under feeder-free conditions, fate specification was initiated with activin A and Wnt3a that triggered engagement into definitive endoderm, followed by priming with fibroblast <em>growth</em> <em>factor</em> <em>1</em>0 (FGF<em>1</em>0) and KAAD-cyclopamine. Addition of retinoic acid, boosted by the pancreatic endoderm inducer indolactam V (ILV), yielded pancreatic progenitors expressing pancreatic and duodenal homeobox <em>1</em> (PDX<em>1</em>), neurogenin 3 (NGN3) and neurogenic <em>differentiation</em> <em>1</em> (NEUROD<em>1</em>) markers. Further guidance, under insulin-like <em>growth</em> <em>factor</em> <em>1</em> (IGF-<em>1</em>), hepatocyte <em>growth</em> <em>factor</em> (HGF) and N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), was enhanced by glucagon-like peptide-<em>1</em> (GLP-<em>1</em>) to generate islet-like cells that expressed pancreas-specific markers including insulin and glucagon. Derived progeny demonstrated sustained expression of PDX<em>1</em>, and functional responsiveness to glucose challenge secreting up to 230 pM of C-peptide. A pancreatogenic cocktail enriched with ILV/GLP-<em>1</em> offers a proficient means to specify human iPS cells into glucose-responsive hormone-producing progeny, refining the development of a personalized platform for islet-like cell generation.

Wnt proteins are cysteine-rich glycosylated proteins named after the Drosophilia Wingless (Wg) and the mouse Int-<em>1</em> genes that play a role in <em>embryonic</em> cell patterning, proliferation, <em>differentiation</em>, orientation, adhesion, survival, and programmed cell death (PCD). Wnt proteins involve at least two intracellular signaling pathways. One pathway controls target gene transcription through beta-catenin, generally referred to as the canonical pathway and a second pathway pertains to intracellular calcium (Ca(2+)) release which is termed the non-canonical or Wnt/ Ca(2+) pathway. The majority of Wnt proteins activate gene transcription through the canonical signaling pathway regulated by pathways that include the Frizzled transmembrane receptor and the co-receptor LRP-5/6, Dishevelled, glycogen synthase kinase-3beta (GSK-3beta), adenomatous polyposis coli (APC), and beta-catenin. In contrast, the non-canonical Wnt signaling pathway has two intracellular signaling cascades that consist of the Wnt/ Ca(2+) pathway with protein kinase C (PKC) and the Wnt/PCP pathway involving Rho/Rac small GTPase and Jun N-terminal kinase (JNK). Through a series of signaling pathways, Wnt proteins modulate cell development, proliferation, and cell fate. In regards to cell survival and fate through PCD, Wnt may be critical for the prevention of tissue pathology that involves cytokine and <em>growth</em> <em>factor</em> control during disorders such as neuropsychiatric disease, retinal disease, and Alzheimer's disease. Elucidation of the vital elements that shape and control the Wnt-Frizzled signaling pathway may provide significant prospects for the treatment of disorders of the nervous system.

The specific mechanisms underlying the restorative effects of adrenal chromaffin grafts in experimental parkinsonism are still obscure. Recent findings indicated an involvement of graft-induced trophic interactions in the course of recovery-related events. Evidence that basic fibroblast <em>growth</em> <em>factor</em> (bFGF), a potent trophic protein for neurons, (<em>1</em>) is present in chromaffin cells (Blottner et al., <em>1</em>989) and (2) exerts trophic activities on <em>embryonic</em> mesencephalic neurons in vitro (Ferrari et al., <em>1</em>989) provided the rationale for administering bFGF in gel foam implants unilaterally to the striatum of <em>1</em>-methyl-4-phenyl-<em>1</em>,2,3,6-tetrahydropyridine (MPTP) lesioned mice. Simultaneous bFGF/MPTP treatment diminished bilaterally the reduction of striatal dopamine (DA) levels observed in cytochrome c/MPTP-treated mice and led to an ipsilateral reappearance of tyrosine hydroxylase (TH)-like immunoreactive fibers, most notably adjacent to the implant, 2 weeks after the surgery. Determinations of TH activities and TH immunoblotting demonstrated that bFGF almost fully reversed the loss of TH activity on either side but restored TH protein more on the ipsilateral than on the contralateral side. Furthermore, <em>differences</em> in dihydroxyphenylacetic acid levels, which were about twice as high on the contralateral side yet still reduced with respect to untreated mice, supported our assumption that the molar TH activity was increased on the untreated side, possibly due to an intrinsic compensatory up-regulation. Delayed administration of bFGF starting 8 d after the MPTP treatment was equally effective with regard to morphological parameters. Our results suggest that bFGF partially prevents the deleterious chemical and morphological consequences of an MPTP-mediated nigrostriatal lesion. Thus, bFGF mimics at least the morphological effects of chromaffin cell grafts to the MPTP-lesioned brain.

Previous studies have shown that haploinsufficiency of the splanchnic and septum transversum mesoderm Forkhead Box (Fox) f<em>1</em> transcriptional <em>factor</em> caused defects in lung and gallbladder development and that Foxf<em>1</em> heterozygous (+/-) mice exhibited defective lung repair in response to injury. In this study, we show that Foxf<em>1</em> is expressed in hepatic stellate cells in developing and adult liver, suggesting that a subset of stellate cells originates from septum transversum mesenchyme during mouse <em>embryonic</em> development. Because liver regeneration requires a transient <em>differentiation</em> of stellate cells into myofibroblasts, which secrete type I collagen into the extracellular matrix, we examined Foxf<em>1</em> +/- liver repair following carbon tetrachloride injury, a known model for stellate cell activation. We found that regenerating Foxf<em>1</em> +/- liver exhibited defective stellate cell activation following CCl(4) liver injury, which was associated with diminished induction of type I collagen, alpha-smooth muscle actin, and Notch-2 protein and resulted in severe hepatic apoptosis despite normal cellular proliferation rates. Furthermore, regenerating Foxf<em>1</em> +/- livers exhibited decreased levels of interferon-inducible protein <em>1</em>0 (IP-<em>1</em>0), delayed induction of monocyte chemoattractant protein <em>1</em> (MCP-<em>1</em>) levels, and aberrantly elevated expression of transforming <em>growth</em> <em>factor</em> beta<em>1</em>. In conclusion, Foxf<em>1</em> +/- mice exhibited abnormal liver repair, diminished activation of hepatic stellate cells, and increased pericentral hepatic apoptosis following CCl(4) injury.

We recently discovered the existence of the oxytocin/oxytocin receptor (OT/OTR) system in the heart. Activation of cardiac OTR stimulates the release of atrial natriuretic peptide (ANP), which is involved in regulation of blood pressure and cell <em>growth</em>. Having observed elevated OT levels in the fetal and newborn heart at a stage of intense cardiomyocyte hyperplasia, we hypothesized a role for OT in cardiomyocyte <em>differentiation</em>. We used mouse P<em>1</em>9 <em>embryonic</em> stem cells to substantiate this potential role. P<em>1</em>9 cells give rise to the formation of cell derivatives of all germ layers. Treatment of P<em>1</em>9 cell aggregates with dimethyl sulfoxide (DMSO) induces <em>differentiation</em> to cardiomyocytes. In this work, P<em>1</em>9 cells were allowed to aggregate from day 0 to day 4 in the presence of 0.5% DMSO, <em>1</em>0(-7) M OT and/or <em>1</em>0(-7) M OT antagonist (OTA), and then cultured in the absence of these <em>factors</em> until day <em>1</em>4. OT alone stimulated the production of beating cell colonies in all 24 independently <em>growing</em> cultures by day 8 of the <em>differentiation</em> protocol, whereas the same result was obtained in cells induced by DMSO only after <em>1</em>2 days. Cells induced with OT exhibited increased ANP mRNA, had abundant mitochondria (i.e., they strongly absorbed rhodamine <em>1</em>23), and expressed sarcomeric myosin heavy chain and dihydropyridine receptor-alpha <em>1</em>, confirming a cardiomyocyte phenotype. In addition, OT as well as DMSO increased OTR protein and OTR mRNA, and OTA completely inhibited the formation of cardiomyocytes in OT- and DMSO-supplemented cultures. These results suggest that the OT/OTR system plays an important role in cardiogenesis by promoting cardiomyocyte <em>differentiation</em>.

In vertebrates, the first haematopoietic stem cells (HSCs) with multi-lineage and long-term repopulating potential arise in the AGM (aorta-gonad-mesonephros) region. These HSCs are generated from a rare and transient subset of endothelial cells, called haemogenic endothelium (HE), through an endothelial-to-haematopoietic transition (EHT). Here, we establish the absolute requirement of the transcriptional repressors GFI<em>1</em> and GFI<em>1</em>B (<em>growth</em> <em>factor</em> independence <em>1</em> and <em>1</em>B) in this unique trans-<em>differentiation</em> process. We first demonstrate that Gfi<em>1</em> expression specifically defines the rare population of HE that generates emerging HSCs. We further establish that in the absence of GFI<em>1</em> proteins, HSCs and haematopoietic progenitor cells are not produced in the AGM, revealing the critical requirement for GFI<em>1</em> proteins in intra-<em>embryonic</em> EHT. Finally, we demonstrate that GFI<em>1</em> proteins recruit the chromatin-modifying protein LSD<em>1</em>, a member of the CoREST repressive complex, to epigenetically silence the endothelial program in HE and allow the emergence of blood cells.

The versatile cytokine transforming <em>growth</em> <em>factor</em> β (TGF-β) regulates cellular <em>growth</em>, <em>differentiation</em>, and migration during <em>embryonic</em> development and adult tissue homeostasis. Activation of TGF-β receptors leads to phosphorylation of Smad2 and Smad3, which oligomerize with Smad4 and accumulate in the nucleus where they recognize gene regulatory regions and orchestrate transcription. Termination of Smad-activated transcription involves Smad dephosphorylation, nuclear export, or ubiquitin-mediated degradation. In an unbiased proteomic screen, we identified poly(ADP-ribose) polymerase-<em>1</em> (PARP-<em>1</em>) as a Smad-interacting partner. PARP-<em>1</em> dissociates Smad complexes from DNA by ADP-ribosylating Smad3 and Smad4, which attenuates Smad-specific gene responses and TGF-β-induced epithelial-mesenchymal transition. Thus, our results identify ADP-ribosylation of Smad proteins by PARP-<em>1</em> as a key step in controlling the strength and duration of Smad-mediated transcription.

Here we provide evidence to show that the platelet-derived <em>growth</em> <em>factor</em> beta receptor is tethered to endogenous G-protein-coupled receptor(s) in human <em>embryonic</em> kidney 293 cells. The tethered receptor complex provides a platform on which receptor tyrosine kinase and G-protein-coupled receptor signals can be integrated to produce more efficient stimulation of the p42/p44 mitogen-activated protein kinase pathway. This was based on several lines of evidence. First, we have shown that pertussis toxin (which uncouples G-protein-coupled receptors from inhibitory G-proteins) reduced the platelet-derived <em>growth</em> <em>factor</em> stimulation of p42/p44 mitogen-activated protein kinase. Second, transfection of cells with inhibitory G-protein alpha subunit increased the activation of p42/p44 mitogen-activated protein kinase by platelet-derived <em>growth</em> <em>factor</em>. Third, platelet-derived <em>growth</em> <em>factor</em> stimulated the tyrosine phosphorylation of the inhibitory G-protein alpha subunit, which was blocked by the platelet-derived <em>growth</em> <em>factor</em> kinase inhibitor, tyrphostin AG <em>1</em>296. We have also shown that the platelet-derived <em>growth</em> <em>factor</em> beta receptor forms a tethered complex with Myc-tagged endothelial <em>differentiation</em> gene <em>1</em> (a G-protein-coupled receptor whose agonist is sphingosine <em>1</em>-phosphate) in cells co-transfected with these receptors. This facilitates platelet-derived <em>growth</em> <em>factor</em>-stimulated tyrosine phosphorylation of the inhibitory G-protein alpha subunit and increases p42/p44 mitogen-activated protein kinase activation. In addition, we found that G-protein-coupled receptor kinase 2 and beta-arrestin I can associate with the platelet-derived <em>growth</em> <em>factor</em> beta receptor. These proteins play an important role in regulating endocytosis of G-protein-coupled receptor signal complexes, which is required for activation of p42/p44 mitogen-activated protein kinase. Thus, platelet-derived <em>growth</em> <em>factor</em> beta receptor signaling may be initiated by G-protein-coupled receptor kinase 2/beta-arrestin I that has been recruited to the platelet-derived <em>growth</em> <em>factor</em> beta receptor by its tethering to a G-protein-coupled receptor(s). These results provide a model that may account for the co-mitogenic effect of certain G-protein-coupled receptor agonists with platelet-derived <em>growth</em> <em>factor</em> on DNA synthesis.

Formation of cartilage during both <em>embryonic</em> development and repair processes involves the <em>differentiation</em> of multipotential mesenchymal cells. The mouse cell line, C3H<em>1</em>0T<em>1</em>/2, has been shown to be multipotential and capable of differentiating into various phenotypes normally derived from <em>embryonic</em> mesoderm, including myocytes, adipocytes and chondrocytes. In this study, we have analyzed the induction of chrondrogenesis in C3H<em>1</em>0T<em>1</em>/2 cells by transforming <em>growth</em> <em>factor</em>-beta (TGF-beta <em>1</em>, human recombinant form). Treatment of high-density micromass cultures of C3H<em>1</em>0T<em>1</em>/2 cells with TGF-beta <em>1</em> resulted in the formation of a three dimensional spheroid structure, which exhibited cartilage-like histology. Extracellular matrix components characteristic of cartilage, type II collagen and cartilage link protein, were demonstrated by immunohistochemistry. TGF-beta <em>1</em> treatment increased collagen synthesis, and immunoblot analysis showed the presence of type II collagen in TGF-beta <em>1</em>-treated micromass cultures, but not in TGF-beta <em>1</em>-treated monolayer cultures nor in untreated cultures. An increase in radioactive sulfate uptake relative to DNA synthesis was also seen in TGF-beta <em>1</em>-treated micromass cultures forming spheroids, indicating the increased synthesis of sulfated proteoglycans. These observations indicated that the spheroids formed are of a cartilaginous nature, and that multipotential C3H<em>1</em>0T<em>1</em>/2 cells, which do not spontaneously enter the chondrogenic pathway, can be induced to undergo cellular <em>differentiation</em> towards chondrogenesis in vitro through culture in a favorable environment.

The CCN family of genes constitutes six members of small secreted cysteine rich proteins, which exists only in vertebrates. The major members of CCN are CCN<em>1</em> (Cyr6<em>1</em>), CCN2 (CTGF), and CCN3 (Nov). CCN4, CCN5, and CCN6 were formerly reported to be in the Wisp family, but they are now integrated into CCN due to the resemblance of their four principal modules: insulin like <em>growth</em> <em>factor</em> binding protein, von Willebrand <em>factor</em> type C, thrombospondin type <em>1</em>, and carboxy-terminal domain. CCNs show a wide and highly variable expression pattern in adult and in <em>embryonic</em> tissues, but most studies have focused on their principal role in osteo/chondrogenesis and vasculo/angiogenesis from the aspect of migration, <em>growth</em>, and <em>differentiation</em> of mesenchymal cells. CCN proteins simultaneously integrate and modulate the signals of integrins, bone morphogenetic protein, vascular endothelial <em>growth</em> <em>factor</em>, Wnt, and Notch by direct binding. However, the priority in the use of the signals is different depending on the cell status. Even the equivalent counterparts show a <em>difference</em> in signal usage among species. It may be that the evolution of the CCN family continues to keep pace with vertebrate evolution itself.

Implantation, a critical step for establishing pregnancy, requires molecular and cellular events resulting in uterine <em>growth</em> and <em>differentiation</em>, blastocyst adhesion, invasion, and placental formation. Successful implantation requires a receptive endometrium, a normal and functional embryo at the blastocyst stage, and a synchronized dialogue between maternal and <em>embryonic</em> tissues. In addition to the well-characterized role of sex steroids, the complexity of embryo implantation and placentation is exemplified by the number of cytokines and <em>growth</em> <em>factors</em> with demonstrated roles in these processes. Disturbances in the normal expression and action of these cytokines result in an absolute or partial failure of implantation and abnormal placental formation in mice and human. Members of the gp<em>1</em>30 cytokine family, interleukin-<em>1</em><em>1</em> (IL-<em>1</em><em>1</em>) and leukemia inhibitory <em>factor</em>, the transforming <em>growth</em> <em>factor</em> beta superfamily, the colony-stimulating <em>factors</em>, and the IL-<em>1</em> and IL-<em>1</em>5 systems are crucial molecules for a successful implantation. Chemokines are also important, both in recruiting specific cohorts of leukocytes to the implantation site and in trophoblast trafficking and <em>differentiation</em>. This review provides discussion of the <em>embryonic</em> and uterine <em>factors</em> that are involved in the process of implantation in autocrine, paracrine, and/or juxtacrine manners at the hormonal, cellular, and molecular levels.

Spred/Sprouty family proteins negatively regulate <em>growth</em> <em>factor</em>-induced ERK activation. Although the individual physiological roles of Spred-<em>1</em> and Spred-2 have been investigated using gene-disrupted mice, the overlapping functions of Spred-<em>1</em> and Spred-2 have not been clarified. Here, we demonstrate that the deletion of both Spred-<em>1</em> and Spred-2 resulted in <em>embryonic</em> lethality at <em>embryonic</em> days <em>1</em>2.5 to <em>1</em>5.5 with marked subcutaneous hemorrhage, edema, and dilated lymphatic vessels filled with erythrocytes. This phenotype resembled that of Syk(-/-) and SLP-76(-/-) mice with defects in the separation of lymphatic vessels from blood vessels. The number of LYVE-<em>1</em>-positive lymphatic vessels and lymphatic endothelial cells increased markedly in Spred-<em>1</em>/2-deficient embryos compared with WT embryos, while the number of blood vessels was not different. Ex vivo colony assay revealed that Spred-<em>1</em>/2 suppressed lymphatic endothelial cell proliferation and/or <em>differentiation</em>. In cultured cells, the overexpression of Spred-<em>1</em> or Spred-2 strongly suppressed vascular endothelial <em>growth</em> <em>factor</em>-C (VEGF-C)/VEGF receptor (VEGFR)-3-mediated ERK activation, while Spred-<em>1</em>/2-deficient cells were extremely sensitive to VEGFR-3 signaling. These data suggest that Spreds play an important role in lymphatic vessel development by negatively regulating VEGF-C/VEGFR-3 signaling.

Although most cells in the <em>embryonic</em> mouse cortex express the serine-threonine kinase Akt-<em>1</em>, a small population of progenitors expresses Akt-<em>1</em> protein at a higher level. To determine the functional significance of this <em>difference</em>, we used a retrovirus to increase Akt-<em>1</em> expression in cortical progenitors. Increased Akt expression enhanced Akt activation after <em>growth</em> <em>factor</em> stimulation of progenitors. In vivo, it promoted retention in progenitor layers, the ventricular zone and subventricular zone. In vitro, it enhanced proliferation and survival, but did not impair migration. Moreover, it increased the proportion of stem cells, defined by a self-renewal assay. These effects did not depend on the Akt substrate p2<em>1</em>(Cip<em>1</em>). In contrast, rapamycin, an inhibitor of mTOR (mammalian target of rapamycin), altered effects of elevated Akt-<em>1</em> selectively: it eliminated the increase in stem cells and reduced the proliferative response, but had no effect on survival. The ability of elevated Akt-<em>1</em> to increase the self-renewing population therefore depends on a rapamycin-sensitive mechanism (presumably inhibition of mTOR activity) but not on p2<em>1</em>(Cip<em>1</em>), and can be distinguished from its effects on the proliferation and survival of other types of progenitors. Our findings suggest that expression of a high level of Akt-<em>1</em> by a subpopulation of cortical progenitors biases their responses to extrinsic signals to increase their survival, proliferation, and/or self-renewal. Heterogeneity in Akt-<em>1</em> level among progenitors could therefore allow cells that share a microenvironment to respond differently to the same extrinsic signals.

Binding sites for transcription <em>factor</em> nuclear <em>factor</em> one (NFI) proteins, encoded by four genes in the mouse, have been characterized from many tissue-specific genes. NFI genes are expressed in unique but overlapping patterns in <em>embryonic</em> and in adult tissues. Nfib is highly expressed in the <em>embryonic</em> lung. Here we show that Nfib null mutants die early postnatally and display severe lung hypoplasia. Heterozygotes do survive, but exhibit delayed pulmonary <em>differentiation</em>. Expression of transforming <em>growth</em> <em>factor</em> beta <em>1</em> (TGF-beta<em>1</em>) and sonic hedgehog (Shh) is not down-regulated in mutant lung epithelium at late stages of morphogenesis, which may result in incomplete lung maturation. Our study demonstrates that Nfib is essential for normal lung development, and suggests that it could be involved in the pathogenesis of respiratory distress syndromes in humans.

In birds and mammals, cardiac myocytes terminate mitotic activity in the neonatal period and regeneration of cardiac muscle does not occur after myocardial injury in adult hearts. Even <em>embryonic</em> myocytes, which actively proliferate in vivo, quickly lose mitotic activity when placed in cell culture. Several <em>growth</em> <em>factors</em>, including fibroblast <em>growth</em> <em>factor</em> (FGF), have been documented in <em>embryonic</em> hearts and some have been shown to influence myocyte terminal <em>differentiation</em> in culture. However, none of these <em>growth</em> <em>factors</em> have been shown to reactivate cell division in postmitotic myocytes nor have their in vivo functions been defined satis<em>factor</em>ily. To clarify the role of FGF signaling in heart <em>growth</em>, we prepared two retroviral vectors capable of suppressing (i) functions of FGF receptors (FGFRs) with a dominant-negative mutant of receptor type <em>1</em> (FGFR<em>1</em>) or (ii) the translation of endogenous FGFR<em>1</em> by transcribing its antisense RNA. Both vectors inhibited myocyte proliferation and/or survival during the first week of chicken <em>embryonic</em> development but had much less effect after the second week. No apparent alteration of myocyte <em>growth</em> was observed after overexpression of full-length FGFR<em>1</em>. These results suggest that receptor-coupled FGF signaling regulates cardiac myocyte <em>growth</em> during tubular stages of cardiogenesis but that myocyte <em>growth</em> becomes FGF-independent after the second week of embryogenesis.

The corneal epithelial basement membrane (BM) is positioned between basal epithelial cells and the stroma. This highly specialized extracellular matrix functions not only to anchor epithelial cells to the stroma and provide scaffolding during <em>embryonic</em> development but also during migration, <em>differentiation</em>, and maintenance of the differentiated epithelial phenotype. Basement membranes are composed of a diverse assemblage of extracellular molecules, some of which are likely specific to the tissue where they function; but in general they are composed of four primary components--collagens, laminins, heparan sulfate proteoglycans, and nidogens--in addition to other components such as thrombospondin-<em>1</em>, matrilin-2, and matrilin-4 and even fibronectin in some BM. Many studies have focused on characterizing BM due to their potential roles in normal tissue function and disease, and these structures have been well characterized in many tissues. Comparatively few studies, however, have focused on the function of the epithelial BM in corneal physiology. Since the normal corneal stroma is avascular and has relatively low keratocyte density, it is expected that the corneal BM would be different from the BM in other tissues. One function that appears critical in homeostasis and wound healing is the barrier function to penetration of cytokines from the epithelium to stroma (such as transforming <em>growth</em> <em>factor</em> β-<em>1</em>), and possibly from stroma to epithelium (such as keratinocyte <em>growth</em> <em>factor</em>). The corneal epithelial BM is also involved in many inherited and acquired corneal diseases. This review examines this structure in detail and discusses the importance of corneal epithelial BM in homeostasis, wound healing, and disease.

In general, human <em>embryonic</em> stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)(<em>1</em>) can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial. The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium(2). Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs(3). Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)(4). Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions(5-7). Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established(8). To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells(9), adult skin cells(<em>1</em>0), foreskin fibroblasts(<em>1</em><em>1</em>-<em>1</em>2), amniotic mesenchymal cells(<em>1</em>3)). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A(<em>1</em>4). Obviously, there is an evident <em>difference</em> in <em>growth</em> <em>factor</em> production by mouse and human feeder cells. Analyses of the transcriptomes of mouse and human feeder cells revealed significant <em>differences</em> between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key <em>factor</em> regulating the expression of Tgfβ<em>1</em>, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs(<em>1</em>5-<em>1</em>6). For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the <em>growth</em> of hESCs. Here, we present an optimized method for the isolation and culture of mouse <em>embryonic</em> fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

Perfluorooctanoic acid (PFOA) is a member of a family of perfluorinated chemicals that have a variety of applications. PFOA persists in the environment and is found in wildlife and humans. In mice, PFOA is developmentally toxic producing mortality, delayed eye opening, <em>growth</em> deficits, and altered pubertal maturation. PFOA activates peroxisome proliferators-activated receptor-alpha (PPARalpha), a pathway critical to the mode of induction of liver tumors in rodents. The present study uses <em>1</em>29S<em>1</em>/SvlmJ wild-type (WT) and PPARalpha knockout (KO) mice to determine if PPARalpha mediates PFOA-induced developmental toxicity. Pregnant mice were dosed orally from gestation days <em>1</em>-<em>1</em>7 with water or 0.<em>1</em>, 0.3, 0.6, <em>1</em>, 3, 5, <em>1</em>0, or 20 mg PFOA/kg. PFOA did not affect maternal weight, <em>embryonic</em> implantation, number, or weight of pups at birth. At 5 mg/kg, the incidence of full litter resorptions increased in both WT and KO mice. In WT, but not KO, neonatal survival was reduced (0.6 mg/kg) and eye opening was delayed (<em>1</em> mg/kg). There was a trend across dose for reduced pup weight (WT and KO) on several postnatal days (PND), but only WT exposed to <em>1</em> mg/kg were significantly different from control (PND7-<em>1</em>0 and 22). Maternal <em>factors</em> (e.g., background genetics) did not contribute to <em>differences</em> in postnatal mortality, as PFOA induced postnatal mortality in heterozygous pups born to WT or KO dams. In conclusion, early pregnancy loss was independent of PPARalpha expression. Delayed eye opening and deficits in postnatal weight gain appeared to depend on PPARalpha expression, although other mechanisms may contribute. PPARalpha was required for PFOA-induced postnatal lethality and expression of one copy of the gene was sufficient to mediate this effect.

Dopamine (DA) neurons can be derived from human and primate <em>embryonic</em> stem (ES) cells in vitro. An ES cell-based replacement therapy for patients with Parkinson's disease requires that in vitro-generated neurons maintain their phenotype in vivo. Other critical issues relate to their proliferative capacity and risk of tumor formation, and the capability of migration and integration in the adult mammalian brain. Neural induction was achieved by coculture of primate parthenogenetic ES cells (Cyno-<em>1</em>) with stromal cells, followed by sequential exposure to midbrain patterning and <em>differentiation</em> <em>factors</em> to favor DA phenotypic specification. Differentiated ES cells were treated with mitomycin C and transplanted into adult immunosuppressed rodents and into a primate (allograft) with out immunosuppression. A small percentage of DA neurons survived in both rodent and primate hosts for the entire term of the study (4 and 7 months, respectively). Other neuronal and glial populations derived from Cyno-<em>1</em> ES cells showed, in vivo, phenotypic characteristics and <em>growth</em> and migration patterns similar to fetal primate transplants, and a majority of cells (>80%) expressed the forebrain transcription <em>factor</em> brain <em>factor</em> <em>1</em>. No teratoma formation was observed. In this study, we demonstrate long-term survival of DA neurons obtained in vitro from primate ES cells. Optimization of <em>differentiation</em>, cell selection, and cell transfer is required for functional studies of ES-derived DA neurons for future therapeutic applications.

In response to stroke, subpopulations of cortical reactive astrocytes proliferate and express proteins commonly associated with neural stem/progenitor cells such as glial fibrillary acidic protein (GFAP) and Nestin. To examine the stem cell-related properties of cortical reactive astrocytes after injury, we generated GFAP-CreER(TM);tdRFP mice to permanently label reactive astrocytes. We isolated cells from the cortical peri-infarct area 3 d after stroke, and cultured them in neural stem cell medium containing epidermal <em>growth</em> <em>factor</em> and basic fibroblast <em>growth</em> <em>factor</em>. We observed tdRFP-positive neural spheres in culture, suggestive of tdRFP-positive reactive astrocyte-derived neural stem/progenitor cells (Rad-NSCs). Cultured Rad-NSCs self-renewed and differentiated into neurons, astrocytes, and oligodendrocytes. Pharmacological inhibition and conditional knock-out mouse studies showed that Presenilin <em>1</em> and Notch <em>1</em> controlled neural sphere formation by Rad-NSCs after stroke. To examine the self-renewal and <em>differentiation</em> potential of Rad-NSCs in vivo, Rad-NSCs were transplanted into <em>embryonic</em>, neonatal, and adult mouse brains. Transplanted Rad-NSCs were observed to persist in the subventricular zone and secondary Rad-NSCs were isolated from the host brain 28 d after transplantation. In contrast with neurogenic postnatal day 4 NSCs and adult NSCs from the subventricular zone, transplanted Rad-NSCs differentiated into astrocytes and oligodendrocytes, but not neurons, demonstrating that Rad-NSCs had restricted <em>differentiation</em> in vivo. Our results indicate that Rad-NSCs are unlikely to be suitable for neuronal replacement in the absence of genetic or epigenetic modification.