Survival and death of epiblast cells during embryonic stem cell derivation revealed by long-term live-cell imaging with an Oct4 reporter system

Despite the broad literature on embryonic stem cells (ESCs), their derivation process remains enigmatic. This may be because of the lack of experimental systems that can monitor this prolonged cellular process. Here we applied a live-cell imaging technique to monitor the process of ESC derivation over 10 days from morula to outgrowth phase using an Oct4/eGFP reporter system. Our imaging reflects the ‘natural’ state of ESC derivation, as the ESCs established after the imaging were both competent in chimeric mice formation and germ-line transmission. Using this technique, ESC derivation in conventional conditions was imaged. After the blastocoel was formed, the intensity of Oct4 signals attenuated in the trophoblast cells but was maintained in the inner cell mass (ICM). Thereafter, the Oct4-positive cells scattered and their number decreased along with apoptosis of the other Oct4-nagative cells likely corresponds to trophoblast and hypoblast cells, and then only the surviving Oct4-positive cells proliferated and formed the colony. All embryos without exception passed through this cell death phase. Importantly, the addition of caspase inhibitor Z-VAD-FMK to the medium dramatically suppressed the loss of Oct4-positive cells and also other embryo-derived cells, suggesting that the cell deaths was induced by a caspase-dependent apoptotic pathway. Next we imaged the ESC derivation in 3i medium, which consists of chemical compounds that can suppress differentiation. The most significant difference between the conventional and 3i methods was that there was no obvious cell death in 3i, so that the colony formation was rapid and all of the Oct4-positive cells contributed to the formation of the outgrown colony. These data indicate that the prevention of cell death in epiblast cells is one of the important events for the successful establishment of ESCs. Thus, our imaging technique can advance the understanding of the time-dependent cellular changes during ESC derivation.     

AmfS, an extracellular peptidic morphogen in Streptomyces griseus

The amf gene cluster was previously identified as a regulator for the onset of aerial-mycelium formation in Streptomyces griseus. The nucleotide sequences of amf and its counterparts in other species revealed a conserved gene organization consisting of five open reading frames. A nonsense mutation in amfS, encoding a 43-amino-acid peptide, caused significant blocking of aerial-mycelium formation and streptomycin production, suggesting its role as a regulatory molecule. Extracellular-complementation tests for the aerial-mycelium-deficient phenotype of the amfS mutant demonstrated that AmfS was secreted by the wild-type strain. A null mutation in amfBA, encoding HlyB-like membrane translocators, abolished the extracellular AmfS activity without affecting the wild-type morphology, which suggests that AmfBA is involved not in production but in export of AmfS. A synthetic C-terminal octapeptide partially induced aerial-mycelium formation in the amfS mutant, which suggests that an AmfS derivative, but not AmfS itself, serves as an extracellular morphogen.     

Genome-wide transcriptional orchestration of circadian rhythms in Drosophila

Circadian rhythms govern the behavior, physiology, and metabolism of living organisms. Recent studies have revealed the role of several genes in the clock mechanism both in Drosophila and in mammals. To study how gene expression is globally regulated by the clock mechanism, we used a high density oligonucleotide probe array (GeneChip) to profile gene expression patterns in Drosophila under light-dark and constant dark conditions. We found 712 genes showing a daily fluctuation in mRNA levels under light-dark conditions, and among these the expression of 115 genes was still cycling in constant darkness, i.e. under free-running conditions. Unexpectedly the expression of a large number of genes cycled exclusively under constant darkness. We found that cycling in most of these genes was lost in the arrhythmic Clock (Clk) mutant under light-dark conditions. Expression of periodically regulated genes is coordinated locally on chromosomes where small clusters of genes are regulated jointly. Our findings reveal that many genes involved in diverse functions are under circadian control and reveal the complexity of circadian gene expression in Drosophila.     

A putative met-enkephalin releaser, kyotorphin enhances intracellular Ca2+ in the synaptosomes

Kyotorphin (Tyr-Arg) at 1 to 100 microM increased the intracellular [Ca2+]i, determined with Quin-II in the slice and the entry of 45Ca2+ entry into synaptosomes of the lower brain stem of the rat. These effects were not antagonized by nifedipine nor verapamil. However, since this dipeptide caused no changes on the membrane potentials of the synaptosomes, measured with Rhodamine 6G, it is suggested that the kyotorphin-induced increase in the [Ca2+]i may be due not to effects on the voltage dependent Ca2+ channels and Na+-Ca2+ exchange mechanisms caused by the changes of the membrane potentials, but to the specific receptor (kyotorphin receptor)-mediated mechanisms.     

A deletion in a cis element of Foxe3 causes cataracts and microphthalmia in rct mice

The Rinshoken cataract (rct) mutation, which causes congenital cataracts, is a recessive mutation found in SJL/J mice. All mutants present with opacity in the lens by 2?months of age. The rct locus was mapped to a 1.6-Mb region in Chr 4 that contains the Foxe3 gene. This gene is responsible for cataracts in humans and mice, and it plays a crucial role in the development of the lens. Furthermore, mutation of Foxe3 causes various ocular defects. We sequenced the genomic region of Foxe3, including the coding exons and UTRs; however, no mutations were discovered in these regions. Because there were no differences in Foxe3 sequences between the rct/rct and wild-type mice, we inferred that a mutation was located in the regulatory regions of the Foxe3 gene. To test this possibility, we sequenced a 5' noncoding region that is highly conserved among vertebrates and is predicted to be the major enhancer of Foxe3. This analysis revealed a deletion of 22-bp located approximately 3.2-kb upstream of the start codon of Foxe3 in rct mice. Moreover, we demonstrated by RT-PCR and in situ hybridization that the rct mutant has reduced expression of Foxe3 in the lens during development. We therefore suggest that cataracts in rct mice are caused by reduced Foxe3 expression in the lens and that this decreased expression is a result of a deletion in a cis-acting regulatory element.     

Studies on the ontogenesis and the regulatory mechanisms of placental lactogen and insulin binding sites (receptor) in rat liver

The ontogenesis of specific binding of 125I-hPL and 125I-insulin was determined in rat liver cell membranes (10(5) X g pellets), and the regulatory mechanisms of these binding sites were also examined. There were striking differences in the mode of ontogenesis between binding sites of hPL and insulin in rats. HPL binding sites were very few in liver cell membranes from fetal and immature rats. They began to increase after puberty, and markedly increased in late pregnancy. On the other hand, insulin binding sites, which decreased in late pregnancy, were dominant in fetal liver and placenta. Consequently, the lipolytic and glycogenolytic activities of hPL in maternal liver were accentuated, whereas the effects of insulin on maternal liver were suppressed. In contrast, in fetal liver and placenta only the anabolic effects of insulin seemed conspicuous. According to the results of experiments on in vivo administration of estradiol-17 beta, progesterone, hydrocortisone or hPL to intact or hypox-rats, and the measurement of serum rat chorionic mammotropin (rCM), rPRL, estradiol-17 beta, and insulin during pregnancy in rats, the increase in hepatic hPL binding sites observed in late pregnancy might be, at least in part, due to rCM secreted from placenta, and the decrease in insulin binding sites due to the increase in serum insulin itself in rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Down-regulation of the mitochondrial translation system during terminal differentiation of HL-60 cells by 12-O-tetradecanoyl-1-phorbol-13-acetate: comparison with the cytoplasmic translation system

Mitochondrial (mt) biogenesis depends on both the nuclear and mt genomes, and a coordination of these two genetic systems is necessary for proper cell functioning. Little is known about the regulatory mechanisms of mt translation or about the expression of mt translation factors. Here, we studied the expression of mt translation factors during 12-O-tetradecanoyl-1-phorbol-13-acetate (TPA)-induced terminal differentiation of HL-60 cells. For all mt translation factors investigated, mRNA expression was markedly down-regulated in a coordinate and specific manner, whereas mRNA levels for the cytoplasmic translation factors showed only a slight reduction. An actinomycin D chase study and nuclear run-on assay revealed that the TPA-induced decrease in mt elongation factor Tu (EF-Tumt) mRNA mainly results from decreased mRNA stability. Polysome analysis showed that there was no significant translational control of mt translation factor (EF-Tumt, ribosomal proteins L7/L12mt and S12mt) mRNA expression during differentiation. Thus, the decreased protein level of one of these mt translation factors (EF-Tumt) simply reflects its decreased mRNA level. It was also demonstrated by pulse labeling of mt translation products that the down-regulation of mt translational activity is actually associated with down-regulated mt translation factor expression during cellular differentiation. Our results illustrate that the regulatory mechanisms of mt translational activity upon terminal differentiation (in response to the growth arrest) is different to that of the cytoplasmic system, where the control of mRNA translational efficiency of major translation factors is the central mechanism for their down-regulation.