Germ cell fate in mice is induced in pluripotent epiblast cells in response to signals from extraembryonic tissues. The specification of approximately 40 founder primordial germ cells and their segregation from somatic neighbours are important events in early development. We have proposed that a critical event during this specification includes repression of a somatic programme that is adopted by neighbouring cells. Here we show that Blimp1 (also known as Prdm1), a known transcriptional repressor, has a critical role in the foundation of the mouse germ cell lineage, as its disruption causes a block early in the process of primordial germ cell formation. Blimp1-deficient mutant embryos form a tight cluster of about 20 primordial germ cell-like cells, which fail to show the characteristic migration, proliferation and consistent repression of homeobox genes that normally accompany specification of primordial germ cells. Furthermore, our genetic lineage-tracing experiments indicate that the Blimp1-positive cells originating from the proximal posterior epiblast cells are indeed the lineage-restricted primordial germ cell precursors.

Specification of germ cell fate is fundamental in development and heredity. Recent evidence indicates that in mice, specification of primordial germ cells (PGCs), the common source of both oocytes and spermatozoa, occurs through the integration of three key events: repression of the somatic program, reacquisition of potential pluripotency and ensuing genome-wide epigenetic reprogramming. Here we provide genetic evidence that Prdm14, a PR domain-containing transcriptional regulator with exclusive expression in the germ cell lineage and pluripotent cell lines, is critical in two of these events, the reacquisition of potential pluripotency and successful epigenetic reprogramming. In Prdm14 mutants, the failure of these two events manifests even in the presence of Prdm1 (also known as Blimp1), a key transcriptional regulator for PGC specification. Our combined evidence demonstrates that Prdm14 defines a previously unknown genetic pathway, initiating independently from Prdm1, for ensuring the launching of the mammalian germ cell lineage.

Epidermal lineage commitment occurs when multipotent stem cells are specified to three lineages: the epidermis, the hair follicle, and the sebaceous gland (SG). How and when a lineage becomes specified remains unknown. Here, we report the existence of a population of unipotent progenitor cells that reside in the SG and express the transcriptional repressor Blimp1. Using cell-culture studies and genetic lineage tracing, we demonstrate that Blimp1-expressing cells are upstream from other cells of the SG lineage. Blimp1 appears to govern cellular input into the gland since its loss leads to elevated c-myc expression, augmented cell proliferation, and SG hyperplasia. Finally, BrdU labeling experiments demonstrate that the SG defects associated with loss of Blimp1 lead to enhanced bulge stem cell activity, suggesting that when normal SG homeostasis is perturbed, multipotent stem cells in the bulge can be mobilized to correct this imbalance.

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.

Specification of germ cell fate is fundamental in development. With a highly representative single-cell microarray and rigorous quantitative PCR analysis, we defined the genome-wide transcription dynamics that create primordial germ cells (PGCs) from the epiblast, a process that exclusively segregates them from their somatic neighbors. We also analyzed the effect of the loss of Blimp1, a key transcriptional regulator, on these dynamics. Our analysis revealed that PGC specification involves complex, yet highly ordered regulation of a large number of genes, proceeding under the strong influence of mesoderm induction but specifically avoiding developmental programs such as the epithelial-mesenchymal transition, Hox cluster activation, cell cycle progression, and DNA methyltransferase machinery. Remarkably, Blimp1 is essential for repressing nearly all the genes normally down-regulated in PGCs relative to their somatic neighbors. In contrast, it is dispensable for the activation of approximately half of the genes up-regulated in PGCs, uncovering the Blimp1-independent events for PGC specification. Notably, however, highly PGC-specific genes exhibited distinct correlations to Blimp1 in wild-type embryos, and these correlations faithfully predicted their expression impairments in Blimp1 mutants. Moreover, their expression overlaps within single cells were severely damaged without Blimp1, demonstrating that Blimp1 exerts positive influence on their concerted activation. Thus, Blimp1 is not a single initiator but a dominant coordinator of the transcriptional program for the establishment of the germ cell fate in mice.[Keywords: Primordial germ cells; Blimp1/Prdm1; cell fate specification; single-cell analysis; genome-wide gene expression profiling] Supplemental material is available at http://www.genesdev.org.

Specification of the germ cell lineage is vital to development and heredity. In mice, the germ cell fate is induced in pluripotent epiblast cells by signaling molecules, yet the underlying mechanism remains unknown. Here we demonstrate that germ cell fate in the epiblast is a direct consequence of Bmp4 signaling from the extraembryonic ectoderm (ExE), which is antagonized by the anterior visceral endoderm (AVE). Strikingly, Bmp8b from the ExE restricts AVE development, thereby contributing to Bmp4 signaling. Furthermore, Wnt3 in the epiblast ensures its responsiveness to Bmp4. Serum-free, defined cultures revealed that, in response to Bmp4, competent epiblast cells uniformly expressed key transcriptional regulators Blimp1 and Prdm14 and acquired germ-cell properties, including genome-wide epigenetic reprogramming, in an orderly fashion. Notably, the induced cells contributed to both spermatogenesis and fertility of offspring. By identifying a signaling principle in germ cell specification, our study establishes a robust strategy for reconstituting the mammalian germ cell lineage in vitro.

Germ cell fate in mice is induced in proximal epiblast cells at Embryonic Day (E) 6.5 by signaling molecules. Prdm1(also known as Blimp1)-positive lineage-restricted precursors of primordial germ cells (PGCs) initiate the formation of a cluster that differentiates into Dppa3 (also known as stella)-positive PGCs from around E7.0 onwards in the extra-embryonic mesoderm. Around E7.5, these PGCs begin migrating towards the definitive endoderm, with concomitant extensive epigenetic reprogramming. To gain a more precise insight into the mechanism of PGC specification and its subsequent development, we exploited quantitative, single-cell, gene expression profiling to explore gene expression dynamics during the 36 h of PGC differentiation from E6.75 to E8.25, in comparison with the corresponding profiles of somatic neighbors. This analysis revealed that the transitions from Prdm1-positive PGC precursors to Dppa3-positive PGCs and to more advanced migrating PGCs involve a highly dynamic, stage-dependent transcriptional orchestration that begins with the regaining of the pluripotency-associated gene network, followed by stepwise activation of PGC-specific genes, differential repression of the somatic mesodermal program, as well as potential modulations of signal transduction capacities and unique control of epigenetic regulators. The information presented here regarding the cascade of events involved in PGC development should serve as a basis for detailed functional analyses of the gene products associated with this process, as well as for appropriate reconstitution of PGCs and their descendant cells in culture.

We describe here a protocol for the representative amplification of global mRNAs from typical single mammalian cells to provide a template for high-density oligonucleotide microarray analysis. A single cell is lysed in a tube without purification and first-strand cDNAs are synthesized using a poly(dT)-tailed primer. Unreacted primer is specifically eliminated by exonuclease treatment and second strands are generated with a second poly(dT)-tailed primer after poly(dA) tailing of the first-strand cDNAs. The cDNAs are split into four tubes, which are independently directionally amplified by PCR, and then recombined. The amplified products (approximately 100 ng) show superior representation and reproducibility of original gene expression, especially for genes expressed in more than 20 copies per cell, compared with those obtained by a conventional PCR protocol, and can effectively be used for quantitative PCR and EST analyses. The cDNAs are then subjected to another PCR amplification with primers bearing the T7 promoter sequence. The resultant cDNA products are gel purified, amplified by one final cycle and used for isothermal linear amplification by T7 RNA polymerase to synthesize cRNAs for microarray hybridization. This protocol yields cDNA templates sufficient for more than 80 microarray hybridizations from a single cell, and can be completed in 5-6 days.