Next-generation sequencing technology is a powerful tool for transcriptome analysis. However, under certain conditions, only a small amount of material is available, which requires more sensitive techniques that can preferably be used at the single-cell level. Here we describe a single-cell digital gene expression profiling assay. Using our mRNA-Seq assay with only a single mouse blastomere, we detected the expression of 75% (5,270) more genes than microarray techniques and identified 1,753 previously unknown splice junctions called by at least 5 reads. Moreover, 8-19% of the genes with multiple known transcript isoforms expressed at least two isoforms in the same blastomere or oocyte, which unambiguously demonstrated the complexity of the transcript variants at whole-genome scale in individual cells. Finally, for Dicer1(-/-) and Ago2(-/-) (Eif2c2(-/-)) oocytes, we found that 1,696 and 1,553 genes, respectively, were abnormally upregulated compared to wild-type controls, with 619 genes in common.

Measuring gene expression in individual cells is crucial for understanding the gene regulatory network controlling human embryonic development. Here we apply single-cell RNA sequencing (RNA-Seq) analysis to 124 individual cells from human preimplantation embryos and human embryonic stem cells (hESCs) at different passages. The number of maternally expressed genes detected in our data set is 22,687, including 8,701 long noncoding RNAs (lncRNAs), which represents a significant increase from 9,735 maternal genes detected previously by cDNA microarray. We discovered 2,733 novel lncRNAs, many of which are expressed in specific developmental stages. To address the long-standing question whether gene expression signatures of human epiblast (EPI) and in vitro hESCs are the same, we found that EPI cells and primary hESC outgrowth have dramatically different transcriptomes, with 1,498 genes showing differential expression between them. This work provides a comprehensive framework of the transcriptome landscapes of human early embryos and hESCs.

Summary Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and non-tumorigenic cancer cells. Three clusters, miR-200c-141, miR-200b-200a-429 and miR-183-96-182 were down-regulated in human BCSCs, normal human and murine mammary stem/progenitor cells and embryonal carcinoma cells. Expression of BMI1, a known regulator of stem cell self-renewal, was modulated by miR-200c. MiR-200c inhibited the clonogenicity of breast cancer cells and suppressed the growth of embryonal carcinoma cells in vitro. Most importantly, miR-200c strongly suppressed the ability of normal mammary stem cells to form mammary ducts and tumor formation driven by human BCSCs in vivo. The coordinated down-regulation of three microRNA clusters and the similar functional regulation of clonogenicity by miR-200c provide a molecular link that connects breast cancer stem cells with normal stem cells.

We directly observed real-time production of single protein molecules in individual Escherichia coli cells. A fusion protein of a fast-maturing yellow fluorescent protein (YFP) and a membrane-targeting peptide was expressed under a repressed condition. The membrane-localized YFP can be detected with single-molecule sensitivity. We found that the protein molecules are produced in bursts, with each burst originating from a stochastically transcribed single messenger RNA molecule, and that protein copy numbers in the bursts follow a geometric distribution. The quantitative study of low-level gene expression demonstrates the potential of single-molecule experiments in elucidating the workings of fundamental biological processes in living cells.

MicroRNAs (miRNAs) have important roles in diverse cellular processes, but little is known about their identity and functions during early mammalian development. Here, we show the effects of the loss of maternal inheritance of miRNAs following specific deletion of Dicer from growing oocytes. The mutant mature oocytes were almost entirely depleted of all miRNAs, and they failed to progress through the first cell division, probably because of disorganized spindle formation. By comparing single-cell cDNA microarray profiles of control and mutant oocytes, our data are compatible with the notion that a large proportion of the maternal genes are directly or indirectly under the control of miRNAs, which demonstrates that the maternal miRNAs are essential for the earliest stages of mouse embryonic development.Supplemental material is available at http://www.genesdev.org.Received November 20, 2006; revised version accepted January 29, 2007. MicroRNAs (miRNAs) are a large family of short noncoding RNAs (17-25 nucleotides) (He and Hannon 2004). A key function of miRNAs is to repress expression of their target genes through sequence complementation, which reduces the abundance of the target mRNAs and/ or inhibits their translation (Bartel 2004;Bagga et al. 2005). MiRNA genes are first transcribed into miRNA primary transcripts by RNA polymerase II (Kim 2005). These primary transcripts are then processed into miRNA precursors by the Drosha/DGCR8 complex and transported from the nucleus to the cytoplasm. Finally, Dicer processes the miRNA precursors into mature miRNAs. From previous studies, Dicer seems to be critical for early mouse development since its loss of function is embryonic lethal at embryonic day 7.5 (E7.5) (Bernstein et al. 2003).In this study, we have examined the role of miRNAs in the mouse oocyte. The mature oocyte contains a number of molecules that are manufactured during oocyte maturation and utilized during early stages of development before activation of the embryonic genome (Dean 2002). It is likely that miRNAs would also be present in the oocyte, but no information is yet available in the mouse. The purpose of this study was to determine if there is significant inheritance of maternal miRNAs in mammalian zygotes, and to investigate if they play a critical role in early mammalian development. We have investigated how the loss of Dicer affects synthesis of miRNA during oocyte maturation and their impact on mRNA and early development. Results and DiscussionFirst, we decided to investigate if there is significant biogenesis of miRNAs in developing oocytes, and their inheritance in the zygote. We therefore examined expression of miRNAs in single cells during oogenesis by a real-time PCR-based miRNA expression profiling method that we recently developed (C. Tang et al. 2006a,b). We compared the miRNA expression profiles of growing oocytes obtained from females 15-16 d after birth (postnatal days 15-16 [P15-P16]) and at P20-P21, and of mature oocytes from adult females. This analysis reveled dynamic chan...

Capillary electrophoresis systems with integrated electrochemical detection have been microfabricated on glass substrates. Photolithographic placement of the working electrode just outside the exit of the electrophoresis channel provides high-sensitivity electrochemical detection with minimal interference from the separation electric field. Microchip electrophoretic separations of neurotransmitters in under 100 s exemplify the good resolution and attomole detection sensitivity of these devices. Using indirect electrochemical detection, high-sensitivity DNA restriction fragment and PCR product sizing has also been performed. These microdevices match the detector's size to that of microfabricated separation and reaction devices, bringing to reality the lab-on-a-chip concept.

SummaryDuring the transition from the inner cell mass (ICM) cells of blastocysts to pluripotent embryonic stem cells (ESCs) in vitro, a normal developmental program is replaced in cells that acquire a capacity for infinite self-renewal and pluripotency. We explored the underlying mechanism of this switch by using RNA-Seq transcriptome analysis at the resolution of single cells. We detected significant molecular transitions and major changes in transcript variants, which include genes for general metabolism. Furthermore, the expression of repressive epigenetic regulators increased with a concomitant decrease in gene activators that might be necessary to sustain the inherent plasticity of ESCs. Furthermore, we detected changes in microRNAs (miRNAs), with one set that targets early differentiation genes while another set targets pluripotency genes to maintain the unique ESC epigenotype. Such genetic and epigenetic events may contribute to a switch from a normal developmental program in adult cells during the formation of diseased tissues, including cancers.

BackgroundMicroRNAs (miRNAs) are critical regulators of transcriptional and post-transcriptional gene silencing, which are involved in multiple developmental processes in many organisms. Apart from miRNAs, mouse germ cells express another type of small RNA, piwi-interacting RNAs (piRNAs). Although it has been clear that piRNAs play a role in repression of retrotransposons during spermatogenesis, the function of miRNA in mouse germ cells has been unclear.Methodology/Principal FindingsIn this study, we first revealed the expression pattern of miRNAs by using a real-time PCR-based 220-plex miRNA expression profiling method. During development of germ cells, miR-17-92 cluster, which is thought to promote cell cycling, and the ES cell-specific cluster encoding miR-290 to -295 (miR-290-295 cluster) were highly expressed in primordial germ cells (PGCs) and spermatogonia. A set of miRNAs was developmentally regulated. We next analysed function of miRNA biogenesis in germ cell development by using conditional Dicer-knockout mice in which Dicer gene was deleted specifically in the germ cells. Dicer-deleted PGCs and spermatogonia exhibited poor proliferation. Retrotransposon activity was unexpectedly suppressed in Dicer-deleted PGCs, but not affected in the spermatogonia. In Dicer-deleted testis, spermatogenesis was retarded at an early stage when proliferation and/or early differentiation. Additionally, we analysed spermatogenesis in conditional Argonaute2-deficient mice. In contrast to Dicer-deficient testis, spermatogenesis in Argonaute2-deficient testis was indistinguishable from that in wild type.Conclusion/SignificanceThese results illustrate that miRNAs are important for the proliferation of PGCs and spermatogonia, but dispensable for the repression of retrotransposons in developing germ cells. Consistently, miRNAs promoting cell cycling are highly expressed in PGCs and spermatogonia. Furthermore, based on normal spermatogenesis in Argonaute2-deficient testis, the critical function of Dicer in spermatogenesis is independent of Argonaute2.