The retinoblastoma protein, pRB, and the closely related proteins p107 and p130 are important regulators of the mammalian cell cycle. Biochemical and genetic studies have demonstrated overlapping as well as distinct functions for the three proteins in cell cycle control and mouse development. However, the role of the pRB family as a whole in the regulation of cell proliferation, cell death, or cell differentiation is not known. We generated embryonic stem (

Neuronal differentiation is a complex process that involves a plethora of regulatory steps. To identify transcription factors that influence neuronal differentiation we developed a high throughput screen using embryonic stem (ES) cells. Seven-hundred human transcription factor clones were stably introduced into mouse ES (mES) cells and screened for their ability to induce neuronal differentiation of mES cells. Twenty-four factors that are capable of inducing neuronal differentiation were identified, including four known effectors of neuronal differentiation, 11 factors with limited evidence of involvement in regulating neuronal differentiation, and nine novel factors. One transcription factor, Oct-2, was studied in detail and found to be a bifunctional regulator: It can either repress or induce neuronal differentiation, depending on the particular isoform. Ectopic expression experiments demonstrate that isoform Oct-2.4 represses neuronal differentiation, whereas Oct-2.2 activates neuron formation. Consistent with a role in neuronal differentiation, Oct-2.2 expression is induced during differentiation, and cells depleted of Oct-2 and its homolog Oct-1 have a reduced capacity to differentiate into neurons. Our results reveal a number of transcription factors potentially important for mammalian neuronal differentiation, and indicate that Oct-2 may serve as a binary switch to repress differentiation in precursor cells and induce neuronal differentiation later during neuronal development.[Keywords: Oct-2; neuronal; embryonic stem cell; differentiation; high throughput] Supplemental material is available at http://www.genesdev.org. Neuronal development and differentiation are complex processes that involve extracellular factors, local niche, and intracellular factors. Much is known about extracellular factors that mediate neural induction. For example, inhibition of bone morphogenetic protein (BMP) by chordin, noggin (Sasai et al. 1995;Zimmerman et al. 1996), and follistatin (Hemmati-Brivanlou et al. 1994;Fainsod et al. 1997) (Kageyama et al. 2008). In spite of the large number of transcription factors implicated in neuronal development and differentiation, few systematic studies of these regulators have been performed, and thus it is likely that many other transcription factors regulate neuronal differentiation.The complete sequencing of the human genome offers new approaches to systematically identify components important for neuronal differentiation. Gray et al. (2004) examined the expression of most mouse transcription factors using in situ hybridization, and found that at least 20% of known transcription factors have spatially restricted expression patterns in developing mouse brains. While this study provided a wealth of information on the potential involvement of factors in neurogenesis, it lacked any functional information. In principle, another approach that could be developed is to inactivate transcription factors on a large scale; however, for many Cold Spring Harbor Laboratory Press on May 11, 2018 -Publis...

Historically, microbes from the environment have been a reliable source for novel bio-active compounds. Cloning and expression of metagenomic DNA in heterologous strains of bacteria has broadened the range of potential compounds accessible. However, such metagenomic libraries have been under-exploited for applications in mammalian cells because of a lack of integrated methods. We present an innovative platform to systematically mine natural resources for pro-apoptotic compounds that relies on the combination of bacterial delivery and droplet microfluidics. Using the violacein operon from C. violaceum as a model, we demonstrate that E. coli modified to be invasive can serve as an efficient delivery vehicle of natural compounds. This approach permits the seamless screening of metagenomic libraries with mammalian cell assays and alleviates the need for laborious extraction of natural compounds. In addition, we leverage the unique properties of droplet microfluidics to amplify bacterial clones and perform clonal screening at high-throughput in place of one-compound-per-well assays in multi-well format. We also use droplet microfluidics to establish a cell aggregate strategy that overcomes the issue of background apoptosis. Altogether, this work forms the foundation of a versatile platform to efficiently mine the metagenome for compounds with therapeutic potential.

Murine embryonic stem cells were first derived almost 30 years ago from cultured blastocysts and have been primarily used as a tool to better understand development through targeted gene deletions. Only recently has the focus has shifted toward embryonic stem cells themselves and the molecular mechanisms by which they choose a specific cell fate. Through rapid advances in cell culture and genomic modification techniques researchers are beginning to regularly utilize embryonic stem cells for in vitro gene function assays. More important, the mechanisms critical for establishing the pluripotent sate of embryonic stem cells have been elucidated to the point that clinically beneficial stem cell-like counterparts can now be generated from nonembryonic sources. Keywords 1 Embryonic Stem Cells HistoryThe events that lead to the research resulting in the isolation of the first mouse and human embryonic stem cells began in 1953 at the Jackson Laboratory in Bar Harbor, Maine. This initial research was performed by Leroy Stevens, who was funded by a tobacco company determined to prove that the harmfulness of cigarettes stemmed from the wrapping paper and not the tobacco. Stevens observed that male mice of the 129Sv strain would consistently develop teratomas of the testis [63]. Over the course of almost two decades, Stevens' work showed that cells giving rise to teratomas were even present as early as the blastocyst stage, within the inner cell mass [128]. From that observation, the term "embryonic stem cell" was coined. Teratomas were subsequently used to derive cell lines with the ability to differentiate into several cell types [80]. However, because these cell lines had abnormal properties (e.g., abnormal ploidy) and retained the ability to generate teratomas, they were named "embryonic carcinoma" cells. Steven's groundbreaking discovery that pluripotent stem cells existed in early embryos resulted in multiple laboratories determining the proper culture conditions that would allow the propagation of mouse [33, 7 ] embryonic stem (ES) cells.From the onset of culturing murine ES cells, it became apparent that having the technology to introduce heritable genetic changes in the germline would be a powerful asset to studying development and disease in the mouse. Because of that fact, the focus of embryonic stem cell research in the 1980s was not the basic biology of stem cells; instead, the greatest achievement of the ES cell field at that time was considered to be the advent of gene targeting and germline modification [26, 2]. Research conducted along these lines by Mario Capecchi, Martin Evans, and Medicine.A massive resurgence of interest in the properties of ES cells took place in the late 1990s after the efforts of John Hearn's laboratory resulted first in the isolation of ES cells from rhesus monkey and marmoset ([ 5], Table 1) and subsequently to the derivation of human ES (hES) cell lines [ 3].Surprisingly, even though mouse ES cells were first isolated in 1981, rat ES proved to be much more difficult to...