A systems-level understanding of a small but essential population of cells in development or adulthood (e.g. somatic stem cells) requires accurate quantitative monitoring of genome-wide gene expression, ideally from single cells. We report here a strategy to globally amplify mRNAs from single cells for highly quantitative high-density oligonucleotide microarray analysis that combines a small number of directional PCR cycles with subsequent linear amplification. Using this strategy, both the representation of gene expression profiles and reproducibility between individual experiments are unambiguously improved from the original method, along with high coverage and accuracy. The immediate application of this method to single cells in the undifferentiated inner cell masses of mouse blastocysts at embryonic day (E) 3.5 revealed the presence of two populations of cells, one with primitive endoderm (PE) expression and the other with pluripotent epiblast-like gene expression. The genes expressed differentially between these two populations were well preserved in morphologically differentiated PE and epiblast in the embryos one day later (E4.5), demonstrating that the method successfully detects subtle but essential differences in gene expression at the single-cell level among seemingly homogeneous cell populations. This study provides a strategy to analyze biophysical events in medicine as well as in neural, stem cell and developmental biology, where small numbers of distinctive or diseased cells play critical roles.

Direct evidence for the requirement of transcriptional feedback repression in circadian clock function has been elusive. Here, we developed a molecular genetic screen in mammalian cells to identify mutants of the circadian transcriptional activators CLOCK and BMAL1, which were uncoupled from CRYPTOCHROME (CRY)-mediated transcriptional repression. Notably, mutations in the PER-

T he circadian clock is a molecular mechanism underlying endogenous, self-sustained oscillations with a period of Ϸ24 h, manifested in diverse physiological and metabolic processes (1-3). The most striking feature of circadian clock is its flexible yet robust response to various environmental conditions. For example, circadian periodicity varies with light intensity (4-6) while remaining robust over a wide range of temperatures (''temperature compensation'') (1, 3, 7-9). This flexible-yetrobust characteristic is evolutionarily conserved in organisms ranging from photosynthetic bacteria to warm-blooded mammals (3, 10-12), and has interested researchers from a broad range of disciplines. However, despite many genetic and molecular studies (13-22), the detailed biochemical mechanism underlying this characteristic remains poorly elucidated (3).The simplest explanation for this flexible-yet-robust property is that the key period-determining reactions are insensitive to temperature but responsive to other environmental conditions. Indeed, Pittendrigh proposed the existence of a temperatureinsensitive component in the clock system in 1954 (7), and in 1968, he and his colleagues demonstrated that both the wave form and the period of circadian oscillations are invariant with temperature (23). However, the idea of a temperatureinsensitive biochemical reaction is counterintuitive, as elementary chemical processes are highly temperature-sensitive. One exception is the cyanobacterial clock, in which temperatureinsensitive enzymatic reactions are observed (24,25). However, the cyanobacterial clock is quite distinct from other clock systems, and this biochemical mechanism has not been demonstrated in other clocks.Recently, a chemical-biological approach was proposed to help elucidate the basic processes underlying circadian clocks (26), and high-throughput screening of a large chemical compound library was performed (27). In this report, to analyze systematically the fundamental processes involved in determining the period length of mammalian clocks, we tested 1,260 pharmacologically active compounds for their effect on period length in mouse and human clock cell lines, and found 10 compounds that most markedly lengthened the period of both clock cell lines affected both the central and peripheral circadian clocks. Most compounds inhibited CKI or CKI␦ activity, suggesting that CKI /␦-dependent phosphorylation is an important period-determining process in the mammalian circadian clock. Surprisingly, the degradation rate of endogenous PER2, which is regulated by CKI -dependent phosphorylation (28) and probably by CKI␦-dependent phosphorylation, was temperatureinsensitive in the living clock cells, and the temperatureinsensitivity was preserved even for the in vitro CKI /␦-dependent phosphorylation of a synthetic peptide derived from PER2. These results suggest that this period-determining process is flexible in response to chemical perturbation yet robust in the face of temperature perturbations. Based on these findings, we prop...

Direct evidence for the requirement of delay in feedback repression in the mammalian circadian clock has been elusive. Cryptochrome 1 (Cry1), an essential clock component, displays evening-time expression and serves as a strong repressor at morning-time elements (E box/E' box). In this study, we reveal that a combination of day-time elements (D box) within the Cry1-proximal promoter and night-time elements (RREs) within its intronic enhancer gives rise to evening-time expression. A synthetic composite promoter produced evening-time expression, which was further recapitulated by a simple phase-vector model. Of note, coordination of day-time with night-time elements can modulate the extent of phase delay. A genetic complementation assay in Cry1(-/-):Cry2(-/-) cells revealed that substantial delay of Cry1 expression is required to restore circadian rhythmicity, and its prolonged delay slows circadian oscillation. Taken together, our data suggest that phase delay in Cry1 transcription is required for mammalian clock function.

The Drosophila circadian clock consists of integrated autoregulatory feedback loops, making the clock difficult to elucidate without comprehensively identifying the network components in vivo. Previous studies have adopted genome-wide screening for clock-controlled genes using high-density oligonucleotide arrays that identified hundreds of clock-controlled genes. In an attempt to identify the core clock genes among these candidates, we applied genome-wide functional screening using an RNA interference (RNAi) system in vivo. Here we report the identification of novel clock gene candidates including clockwork orange (cwo), a transcriptional repressor belonging to the basic helix-loop-helix ORANGE family. cwo is rhythmically expressed and directly regulated by CLK-CYC through canonical E-box sequences. A genome-wide search for its target genes using the Drosophila genome tiling array revealed that cwo forms its own negative feedback loop and directly suppresses the expression of other clock genes through the E-box sequence. Furthermore, this negative transcriptional feedback loop contributes to sustaining a high-amplitude circadian oscillation in vivo. Based on these results, we propose that the competition between cyclic CLK-CYC activity and the adjustable threshold imposed by CWO keeps E-box-mediated transcription within the controllable range of its activity, thereby rendering a Drosophila circadian clock capable of generating high-amplitude oscillation.