Embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocysts grow infinitely while maintaining pluripotency. Leukemia inhibitory factor (LIF) can maintain self-renewal of mouse ES cells through activation of Stat3. However, LIF/Stat3 is dispensable for maintenance of ICM and human ES cells, suggesting that the pathway is not fundamental for pluripotency. In search of a critical factor(s) that underlies pluripotency in both ICM and ES cells, we performed in silico differential display and identified several genes specifically expressed in mouse ES cells and preimplantation embryos. We found that one of them, encoding the homeoprotein Nanog, was capable of maintaining ES cell self-renewal independently of LIF/Stat3. nanog-deficient ICM failed to generate epiblast and only produced parietal endoderm-like cells. nanog-deficient ES cells lost pluripotency and differentiated into extraembryonic endoderm lineage. These data demonstrate that Nanog is a critical factor underlying pluripotency in both ICM and ES cells.

The clinical features among the affected patients were nearly identical, consisting of relatively mild CNS manifestations and complete recovery within 1 month.

TOR is a serine-threonine kinase that was originally identified as a target of rapamycin in Saccharomyces cerevisiae and then found to be highly conserved among eukaryotes. In Drosophila melanogaster, inactivation of TOR or its substrate, S6 kinase, results in reduced cell size and embryonic lethality, indicating a critical role for the TOR pathway in cell growth control. However, the in vivo functions of mammalian TOR (mTOR) remain unclear. In this study, we disrupted the kinase domain of mouse mTOR by homologous recombination. While heterozygous mutant mice were normal and fertile, homozygous mutant embryos died shortly after implantation due to impaired cell proliferation in both embryonic and extraembryonic compartments. Homozygous blastocysts looked normal, but their inner cell mass and trophoblast failed to proliferate in vitro. Deletion of the C-terminal six amino acids of mTOR, which are essential for kinase activity, resulted in reduced cell size and proliferation arrest in embryonic stem cells. These data show that mTOR controls both cell size and proliferation in early mouse embryos and embryonic stem cells. TOR (target of rapamycin) was originally identified in two mutantSaccharomyces cerevisiae strains, TOR1-1 and TOR2-1, that are resistant to the growth-inhibiting effect of the immunophilin-immunosuppressant complex FKBP (FK506 binding protein) and rapamycin (17). TOR1 and TOR2 are large proteins (Ϸ280 kDa) and are Ϸ70% identical (26,28 (21, 48). mTOR and other members of this family, including ATM, ATR/FPR, and DNA-PKcs, contain C-terminal regions with high similarity to the catalytic domains of phosphoinositide (PI)-3 kinase and PI-4 kinase (26, 28). However, PIKK members are not lipid kinases but rather function as serine-threonine kinases (4, 20). The PIKK proteins contain a short segment at the extreme C terminus that is essential for protein kinase activity and is not present in PI-3 and PI-4 kinases (51).Cell culture studies have demonstrated that mTOR controls protein synthesis, in part by phosphorylating downstream substrates, including p70 s6 kinase (p70 S6K1 ) (3, 5, 20) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) (4, 5, 13, 15). p70 S6K phosphorylates the 40S ribosomal protein S6 and is proposed to play a crucial role in the translation of 5Ј-terminal oligopyrimidine tract mRNAs, which primarily encode ribosomal proteins and components of the translation apparatus (22, 23). Phosphorylation of 4E-BP1 disrupts its binding to eIF4E, a protein that binds the 5Ј cap structure of mRNA. Released eIF4E then forms a functional translation initiation complex with eIF4G, eIF4A, and eIF3 ribosomes, enhancing translation (29, 45). Inactivation of 4E-BP1 and family proteins has a profound effect on translation of mRNAs with complex 5Ј untranslated regions, which often encode regulatory proteins such as protooncogenes (45). The recent discoveries of a 150-kDa binding partner of mTOR, named raptor (regulatory-associated protein of mTOR) (14,27), and its Saccharomyces cerevisi...

Embryonic stem (ES) cells are derived from mammalian blastocysts and maintain pluripotency, an ability to differentiate into all types of somatic and germ cells (32). Another important property of ES cells is their robust and infinite growth equivalent to tumor cells despite their normal karyotype. ES cells were developed from mouse blastocysts in 1981 (8, 15) and have been extensively used to generate knockout mice. Human ES cells were established in 1998 (33) and are considered promising sources for cell transplantation therapy.POU transcription factor Oct3/4 is expressed specifically in pluripotent cells, including ES cells, early embryos, and germ cells (27,31). Targeted disruption of the Oct3/4 gene in the mouse results in early embryonic lethality (21). The inner cellular mass of Oct3/4-null blastocysts differentiates exclusively into trophoblasts. Furthermore, conditional deletion of Oct3/4 in ES cells leads to spontaneous differentiation into trophectoderm (25), demonstrating that Oct3/4 is essential for selfrenewal of ES cells and mouse early development.Only a few Oct3/4 target genes have been identified. These include FGF-4 (4) and Rex-1 (2), in which Oct3/4 binds to an octamer motif, ATT(T/A)GCAT, located in regulatory elements. In FGF-4, SRY-related transcription factor Sox2 binds to a motif adjacent to the octamer sequence and synergistically activates transcription (5). In Rex-1, hypothetical factor ROX1 functions in a similar manner (2). It is not clear whether synergetic interaction with other transcription factors is common among target genes. Even consensus nucleotide sequences of Oct3/4-binding sites have not been fully determined. For example, the Oct3/4-binding site in UTF1 is one nucleotide different from the octamer sequence (22). Furthermore, it remains largely unknown how Oct3/4 maintains self-renewal of ES cells. Identification of novel Oct3/4 target genes is crucial to answering these questions.In this study, we utilized expression analyses, reporter gene analyses, and a gel mobility shift assay to demonstrate that Fbx15, which encodes an F-box-containing protein (35), is a novel target of Oct3/4. We also performed gene-targeting experiments to study physiological functions of Fbx15 in selfrenewal of ES cells, mouse development, and fertility. 467, 318, 198, 408, 239, 400, 453, 109,16, 523, 161, 483, 258, 264, 419, 529, 327, 411, 417, 418, 399, 196, 271, 255, 495, 101, 98, 351, 416, 321, 251, 412, 379, 549, 329, 265, 449, 328, 516, 320, 436, 427, 297, 366, 390, 315, 228, 277, 292, 284, 285, and 30 (a total of 1,328,835 entries). MATERIALS AND METHODS DigitalCell culture. The RF8 (16), JI (13), CGR8 (20), and MG1.19 (9) ES cell lines were cultured as previously described. Differentiation of ES cells was induced with retinoic acid as previously described (36). NIH 3T3 cells were cultured with Dulbecco's modified Eagle medium (Sigma) containing 10% fetal bovine serum (Sanko Junyaku, Tokyo, Japan) and maintained at 37°C with 5% CO 2 .

Oligodendrocyte precursor cells (OPCs) in the adult brain contribute to white matter homeostasis. After white matter damage, OPCs compensate for oligodendrocyte loss by differentiating into mature oligodendrocytes. However, the underlying mechanisms remain to be fully defined. Here, we test the hypothesis that, during endogenous recovery from white matter ischemic injury, astrocytes support the maturation of OPCs by secreting brain-derived neurotrophic factor (BDNF). For in vitro experiments, cultured primary OPCs and astrocytes were prepared from postnatal day 2 rat cortex. When OPCs were subjected to chemical hypoxic stress by exposing them to sublethal CoCl 2 for 7 d, in vitro OPC differentiation into oligodendrocytes was significantly suppressed. Conditioned medium from astrocytes (astro-medium) restored the process of OPC maturation even under the stressed conditions. When astro-medium was filtered with TrkB-Fc to remove BDNF, the BDNF-deficient astro-medium no longer supported OPC maturation. For in vivo experiments, we analyzed a transgenic mouse line (GFAP cre /BDNF wt/fl ) in which BDNF expression is downregulated specifically in GFAP ϩ astrocytes. Both wild-type (GFAP wt /BDNF wt/fl mice) and transgenic mice were subjected to prolonged cerebral hypoperfusion by bilateral common carotid artery stenosis. As expected, compared with wild-type mice, the transgenic mice exhibited a lower number of newly generated oligodendrocytes and larger white matter damage. Together, these findings demonstrate that, during endogenous recovery from white matter damage, astrocytes may promote oligodendrogenesis by secreting BDNF.

Pigmented neurons in the substantia nigra pars compacta (SNc) and locus coeruleus (LC) show decreased numbers differentially in Parkinson's disease (PD) and multiple system atrophy (MSA). Recent reports have described that fast spin-echo T1-weighted magnetic resonance imaging (MRI) by a 3-tesla machine can visualize neuromelanin-related contrast of the noradrenergic and dopaminergic neurons respectively in the LC and the SNc. Using neuromelanin MRI at 3 T, we investigated possible alterations of these catecholaminergic neurons in 32 PD and 9 MSA patients, and compared the results with those of 23 normal volunteers. The contrast ratio of the LC and SNc was decreased in MSA and PD patients, most prominently in the LC in MSA patients. The contrast ratio of the SNc was correlated with the Hoehn-Yahr stage of PD and the severity of neuroradiological abnormalities in MSA. These results indicate a potential diagnostic value of neuromelanin MRI to distinguish MSA patients from normal and PD patients.

Trophic coupling between cerebral endothelium and their neighboring cells is required for the development and maintenance of blood-brain barrier (BBB) function. Here we report that oligodendrocyte precursor cells (OPCs) secrete soluble factor TGF-β1 to support BBB integrity. Firstly, we prepared conditioned media from OPC cultures and added them to cerebral endothelial cultures. Our pharmacological experiments showed that OPC-conditioned media increased expressions of tight-junction proteins and decreased in vitro BBB permeability by activating TGB-β-receptor-MEK/ERK signaling pathway. Secondly, our immuno-electron microscopic observation revealed that in neonatal mouse brains, OPCs attach to cerebral endothelial cells via basal lamina. And finally, we developed a novel transgenic mouse line that TGF-β1 is knocked down specifically in OPCs. Neonates of these OPC-specific TGF-β1 deficient mice (OPC-specific TGF-β1 partial KO mice: PdgfraCre/Tgfb1flox/wt mice or OPC-specific TGF-β1 total KO mice: PdgfraCre/Tgfb1flox/flox mice) exhibited cerebral hemorrhage and loss of BBB function. Taken together, our current study demonstrates that OPCs increase BBB tightness by upregulating tight junction proteins via TGF-β signaling. Although astrocytes and pericytes are well-known regulators of BBB maturation and maintenance, these findings indicate that OPCs also play a pivotal role in promoting BBB integrity.

No abstract