Mechanisms regulating self-renewal and cell fate decisions in mammalian stem cells are poorly understood. We determined global gene expression profiles for mouse and human hematopoietic stem cells and other stages of the hematopoietic hierarchy. Murine and human hematopoietic stem cells share a number of expressed gene products, which define key conserved regulatory pathways in this developmental system. Moreover, in the mouse, a portion of the genetic program of hematopoietic stem cells is shared with embryonic and neural stem cells. This overlapping set of gene products represents a molecular signature of stem cells.

We present an integrated approach to identify genetic mechanisms that control self-renewal in mouse embryonic stem cells. We use short hairpin RNA (shRNA) loss-of-function techniques to downregulate a set of gene products whose expression patterns suggest self-renewal regulatory functions. We focus on transcriptional regulators and identify seven genes for which shRNA-mediated depletion negatively affects self-renewal, including four genes with previously unrecognized roles in self-renewal. Perturbations of these gene products are combined with dynamic, global analyses of gene expression. Our studies suggest specific biological roles for these molecules and reveal the complexity of cell fate regulation in embryonic stem cells.

The high-dimensional data created by high-throughput technologies require visualization tools that reveal data structure and patterns in an intuitive form. We present PHATE, a visualization method that captures both local and global nonlinear structure using an information-geometric distance between datapoints. We compared PHATE to other tools on a variety of artificial and biological *

In the developing cerebral cortex, neurons are born on a predictable schedule. Here we show in mice that the essential timing mechanism is programmed within individual progenitor cells, and its expression depends solely on cell-intrinsic and environmental factors generated within the clonal lineage. Multipotent progenitor cells undergo repeated asymmetric divisions, sequentially generating neurons in their normal in vivo order: first preplate cells, including Cajal-Retzius neurons, then deep and finally superficial cortical plate neurons. As each cortical layer arises, stem cells and neuroblasts become restricted from generating earlier-born neuron types. Growth as neurospheres or in co-culture with younger cells did not restore their plasticity. Using short-hairpin RNA (shRNA) to reduce Foxg1 expression reset the timing of mid- but not late-gestation progenitors, allowing them to remake preplate neurons and then cortical-plate neurons. Our data demonstrate that neural stem cells change neuropotency during development and have a window of plasticity when restrictions can be reversed.

Nanog, Oct4, and Sox2 are the core regulators of mouse (m)ESC pluripotency. Although their basic importance in human (h)ESCs has been demonstrated, the mechanistic functions are not well defined. Here, we identify general and cell-line-specific requirements for NANOG, OCT4, and SOX2 in hESCs. We show that OCT4 regulates, and interacts with, the BMP4 pathway to specify four developmental fates. High levels of OCT4 enable self-renewal in the absence of BMP4 but specify mesendoderm in the presence of BMP4. Low levels of OCT4 induce embryonic ectoderm differentiation in the absence of BMP4 but specify extraembryonic lineages in the presence of BMP4. NANOG represses embryonic ectoderm differentiation but has little effect on other lineages, whereas SOX2 and SOX3 are redundant and repress mesendoderm differentiation. Thus, instead of being panrepressors of differentiation, each factor controls specific cell fates. Our study revises the view of how self-renewal is orchestrated in hESCs.

The complete genomic sequence of Pseudomonas syringae pv. syringae B728a (Pss B728a) has been determined and is compared with that of P. syringae pv. tomato DC3000 (Pst DC3000). The two pathovars of this economically important species of plant pathogenic bacteria differ in host range and other interactions with plants, with Pss having a more pronounced epiphytic stage of growth and higher abiotic stress tolerance and Pst DC3000 having a more pronounced apoplastic growth habitat. The Pss B728a genome (6.1 Mb) contains a circular chromosome and no plasmid, whereas the Pst DC3000 genome is 6.5 mbp in size, composed of a circular chromosome and two plasmids. Although a high degree of similarity exists between the two sequenced Pseudomonads, 976 protein-encoding genes are unique to Pss B728a when compared with Pst DC3000, including large genomic islands likely to contribute to virulence and host specificity. Over 375 repetitive extragenic palindromic sequences unique to Pss B728a when compared with Pst DC3000 are widely distributed throughout the chromosome except in 14 genomic islands, which generally had lower GC content than the genome as a whole. Content of the genomic islands varies, with one containing a prophage and another the plasmid pKLC102 of Pseudomonas aeruginosa PAO1. Among the 976 genes of Pss B728a with no counterpart in Pst DC3000 are those encoding for syringopeptin, syringomycin, indole acetic acid biosynthesis, arginine degradation, and production of ice nuclei. The genomic comparison suggests that several unique genes for Pss B728a such as ectoine synthase, DNA repair, and antibiotic production may contribute to the epiphytic fitness and stress tolerance of this organism.virulence genes ͉ epiphyte ͉ plant pathogen

Knockout studies have shown that the polycomb gene Bmi-1 is important for postnatal, but not embryonic, neural stem cell (NSC) self-renewal and have identified the cell-cycle inhibitors p16/p19 as molecular targets. Here, using lentiviral-delivered shRNAs in vitro and in vivo, we determined that Bmi-1 is also important for NSC self-renewal in the embryo. We found that neural progenitors depend increasingly on Bmi-1 for proliferation as development proceeds from embryonic through adult stages. Acute shRNA-mediated Bmi-1 reduction causes defects in embryonic and adult NSC proliferation and self-renewal that, unexpectedly, are mediated by a different cell-cycle inhibitor, p21. Gene array analyses revealed developmental differences in Bmi-1-controlled expression of genes in the p21-Rb cell cycle regulatory pathway. Our data therefore implicate p21 as an important Bmi-1 target in NSCs, potentially with stage-related differences. Understanding stage-related mechanisms underlying NSC self-renewal has important implications for development of stem cell-based therapies.

Blood cell production originates from a rare population of multipotent, self-renewing stem cells. A genome-wide gene expression analysis was performed in order to define regulatory pathways in stem cells as well as their global genetic program. Subtracted complementary DNA libraries from highly purified murine fetal liver stem cells were analyzed with bioinformatic and array hybridization strategies. A large percentage of the several thousand gene products that have been characterized correspond to previously undescribed molecules with properties suggestive of regulatory functions. The complete data, available in a biological process-oriented database, represent the molecular phenotype of the hematopoietic stem cell.