Cell fate during development is defined by transcription factors that act as molecular switches to activate or repress specific gene expression programmes. The POU transcription factor Oct-3/4 (encoded by Pou5f1) is a candidate regulator in pluripotent and germline cells and is essential for the initial formation of a pluripotent founder cell population in the mammalian embryo. Here we use conditional expression and repression in embryonic stem (ES) cells to determine requirements for Oct-3/4 in the maintenance of developmental potency. Although transcriptional determination has usually been considered as a binary on-off control system, we found that the precise level of Oct-3/4 governs three distinct fates of ES cells. A less than twofold increase in expression causes differentiation into primitive endoderm and mesoderm. In contrast, repression of Oct-3/4 induces loss of pluripotency and dedifferentiation to trophectoderm. Thus a critical amount of Oct-3/4 is required to sustain stem-cell self-renewal, and up- or downregulation induce divergent developmental programmes. Our findings establish a role for Oct-3/4 as a master regulator of pluripotency that controls lineage commitment and illustrate the sophistication of critical transcriptional regulators and the consequent importance of quantitative analyses.

␥-aminobutyric acid (GABA)ergic neurons in the central nervous system regulate the activity of other neurons and play a crucial role in information processing. To assist an advance in the research of GABAergic neurons, here we produced two lines of glutamic acid decarboxylase-green fluorescence protein (GAD67-GFP) knock-in mouse. The distribution pattern of GFP-positive somata was the same as that of the GAD67 in situ hybridization signal in the central nervous system. We encountered neither any apparent ectopic GFP expression in GAD67-negative cells nor any apparent lack of GFP expression in GAD67-positive neurons in the two GAD67-GFP knock-in mouse lines. The timing of GFP expression also paralleled that of GAD67 expression. Hence, we constructed a map of GFP distribution in the knock-in mouse brain. Moreover, we used the knock-in mice to investigate the colocalization of GFP with NeuN, calretinin (CR), parvalbumin (PV), and somatostatin (SS) in the frontal motor cortex. The proportion of GFP-positive cells among NeuN-positive cells (neocortical neurons) was approximately 19.5%. All the CR-, PV-, and SS-positive cells appeared positive for GFP. The CR-, PV, and SS-positive cells emitted GFP fluorescence at various intensities characteristics to them. The proportions of CR-, PV-, and SS-positive cells among GFP-positive cells were 13.9%, 40.1%, and 23.4%, respectively. Thus, the three subtypes of GABAergic neurons accounted for 77.4% of the GFP-positive cells. They accounted for 6.5% in layer I. In accord with unidentified GFP-positive cells, many mediumsized spherical somata emitting intense GFP fluorescence were observed in layer I.

Endocardial cells are thought to contribute at least in part to the formation of the endocardial cushion mesenchyme. Here, we created Tie2-Cre transgenic mice, in which expression of Cre recombinase is driven by an endothelial-specific promoter/enhancer. To analyze the lineage of Cre expressing cells, we used CAG-CAT-Z transgenic mice, in which expression of lacZ is activated only after Cre-mediated recombination. We detected pan-endothelial expression of the Cre transgene in Tie2-Cre;CAG-CAT-Z double-transgenic mice. This expression pattern is almost identical to Tie2-lacZ transgenic mice. However, interestingly, we observed strong and uniform lacZ expression in mesenchymal cells of the atrioventricular canal of Tie2-Cre;CAG-CAT-Z double-transgenic mice. We also detected lacZ expression in the mesenchymal cells in part of the proximal cardiac outflow tract, but not in the mesenchymal cells of the distal outflow tract and branchial arch arteries. LacZ staining in Tie2-Cre;CAG-CAT-Z embryos is consistent with endocardial-mesenchymal transformation in the atrioventricular canal and outflow tract regions. Our observations are consistent with previously reported results from Cx43-lacZ, Wnt1-Cre;R26R, and Pax3-Cre;R26R transgenic mice, in which lacZ expression in the cardiac outflow tract identified contributions in part from the cardiac neural crest. Tie2-Cre transgenic mice are a new genetic tool for the analyses of endothelial cell-lineage and endothelial cell-specific gene targeting.

Among the nonviral techniques for gene transfer in vivo, the direct injection of plasmid DNA into muscle is simple, inexpensive, and safe. Applications of this method have been limited by the relatively low expression levels of the transferred gene. We investigated the applicability of in vivo electroporation for gene transfer into muscle, using plasmid DNA expressing interleukin-5 (IL-5) as the vector. The tibialis anterior muscles of mice were injected with the plasmid DNA, and then a pair of electrode needles were inserted into the DNA injection site to deliver electric pulses. Five days later, the serum IL-5 levels were assayed. Mice that did not receive electroporation had serum levels of 0.2 ng/ml. Electroporation enhanced the levels to over 20 ng/ml. Histochemical analysis of muscles injected with a lacZ expression plasmid showed that in vivo electroporation increased both the number of muscle fibers taking up plasmid DNA and the copy number of plasmids introduced into the cells. These results demonstrate that gene transfer into muscle by electroporation in vivo is more efficient than simple intramuscular DNA injection.

ATP-sensitive K ؉ (K ATP ) channels regulate many cellular functions by linking cell metabolism to membrane potential. We have generated K ATP channel-deficient mice by genetic disruption of Kir6.2, which forms the K ؉ ion-selective pore of the channel. K ATP channels couple cell metabolism to membrane potential in many tissues (1-7). Classical K ATP channels comprise two subunits: a receptor [SUR1 (8), SUR2A (9), or SUR2B (10)] of sulfonylureas such as glibenclamide and tolbutamide, widely used to treat noninsulin-dependent diabetes mellitus, and an inward rectifier K ϩ channel member, Kir6.2 (11,12). The pancreatic beta cell K ATP channel comprises SUR1 and Kir6.2 (11, 12), while the skeletal muscle and cardiac K ATP channel comprises SUR2A and Kir6.2 (9). The pancreatic beta cell K ATP channels, as ATP and ADP sensors, have been thought to play a critical role in the regulation of glucose-and sulfonylurea-induced insulin secretion (13). In fact, mutations of the SUR1 or Kir6.2 gene are known to cause familial hypoglycemia associated with unregulated insulin secretion (14-18). However, recent studies suggest that both glucose and the sulfonylureas might have additional effects distal to those on the K ATP channels (19-21). In addition, although different roles of the K ATP channels in the various tissues, including cytoprotection in heart and brain ischemia and excitability of muscles and neurons, have been proposed (22,23), no direct evidence has been available. To clarify the physiological roles of K ATP channels in various cellular functions directly, we generated K ATP channel-deficient mice by disruption of the Kir6.2 gene. In the present study, we have focused on the role of the K ATP channels in pancreatic beta cell function. Our data clearly demonstrate that both glucose-and sulfonylureainduced insulin secretion depend critically on K ATP channeldependent pathway, and also suggest that the K ATP channels in skeletal muscle are involved in insulin action. MATERIALS AND METHODSTargeting the Kir6.2 Gene. The Kir6.2 gene was cloned from a 129͞Sv mouse genomic DNA library (Stratagene) by using its cDNA probe. A targeting vector was constructed by inserting the neomycin-resistance gene at the XhoI site in Kir6.2. The herpes simplex virus thymidine kinase gene was inserted downstream (Fig. 1 A). The targeting vector was introduced into E14 embryonic stem (ES) cells by electroporation. The homologous recombinant clone was identified by Southern blot analysis, and homozygous mice (Kir6.2 Ϫ͞Ϫ ) were generated by the standard procedures.Electrophysiology and Measurements of Intracellular Calcium Concentrations ([Ca 2؉ ] i ). Pancreatic islets were isolated by collagenase digestion method (24), and dispersed islet cells were cultured in DMEM supplemented with 10% fetal bovine serum, plated into 3.5-cm dishes containing Cellocate Coverslips (Eppendorf), and incubated at 37°C for 24-72 hr before experiments. The whole-cell recordings, single-channel recordings, and measurements of [Ca 2ϩ ] i in single p...

cAMP is well known to regulate exocytosis in various secretory cells, but the precise mechanism of its action remains unknown. Here, we examine the role of cAMP signaling in the exocytotic process of insulin granules in pancreatic beta cells. Although activation of cAMP signaling alone does not cause fusion of the granules to the plasma membrane, it clearly potentiates both the first phase (a prompt, marked, and transient increase) and the second phase (a moderate and sustained increase) of glucoseinduced fusion events. Interestingly, all granules responsible for this potentiation are newly recruited and immediately fused to the plasma membrane without docking (restless newcomer). Importantly, cAMP-potentiated fusion events in the first phase of glucose-induced exocytosis are markedly reduced in mice lacking the cAMP-binding protein Epac2 (Epac2 ko/ko ). In addition, the small GTPase Rap1, which is activated by cAMP specifically through Epac2 in pancreatic beta cells, mediates cAMP-induced insulin secretion in a protein kinase A-independent manner. We also have developed a simulation model of insulin granule movement in which potentiation of the first phase is associated with an increase in the insulin granule density near the plasma membrane. Taken together, these data indicate that Epac2/Rap1 signaling is essential in regulation of insulin granule dynamics by cAMP, most likely by controlling granule density near the plasma membrane.insulin secretion ͉ total internal reflection fluorescence microscopy ͉ pancreatic beta cell

Extraembryonic endoderm (ExE) is differentiated from the inner cell mass of the late blastocyst-stage embryo to form visceral and parietal endoderm, both of which have an important role in early embryogenesis. The essential roles of Gata-6 and Gata-4 on differentiation of visceral endoderm have been identified by analyses of knockout mice. Here we report that forced expression of either Gata-6 or Gata-4 in embryonic stem (ES) cells is sufficient to induce the proper differentiation program towards ExE. We believe that this is the first report of a physiological differentiation event induced by the ectopic expression of a transcription factor in ES cells.

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