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Background: Pluripotent embryonic stem cells (ESCs) offer great potential for regenerative medicine. However, efficient in vitro generation of specific desired cell types is still a challenge. We previously established that Smad2/3 signaling, essential for endoderm formation, regulates target gene expression by counteracting epigenetic repression mediated by Polycomb Repressive Complex 2 (PRC2). Although this mechanism has been demonstrated during differentiation and reprogramming, little is known of its role in pluripotent cells., Results: Chromatin immunoprecipitation-deep sequencing of undifferentiated mouse ESCs inhibited for Smad2/3 signaling identified Prdm14, important for protecting pluripotency, as a target gene. Although Prdm14 accumulates the normally repressive PRC2 deposited histone modification H3K27me3 under these conditions, surprisingly, expression increases. Analysis indicates that increased H3K27me3 leads to increased binding of PRC2 accessory component Jarid2 and recruitment of RNA polymerase II. Similar increases were found at the Nodal endoderm target gene Eomes but it remained unexpressed in pluripotent cells as normal. Upon differentiation, however, Eomes expression was significantly higher than in cells that had not been inhibited for signaling before differentiation. In addition, endoderm formation was markedly increased., Conclusions: Blocking Smad2/3 signaling in pluripotent stem cells results in epigenetic changes that enhance the capacity for endoderm differentiation. Developmental Dynamics 245:807-815, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: Human PAX-Interacting Protein 1 (PAXIP1)-associated glutamate rich protein 1 (PAGR1, also known as PA1) originally was discovered as part of a complex containing PAXIP1 and histone H3K4 methyltransferases MLL3 and MLL4, suggesting a role in epigenetic gene regulation. Further in vitro studies suggested additional functions in DNA damage repair and transcription. However, in vivo analysis of PAGR1 function has been lacking., Results: Here we show that expression of the cognate mouse gene Pagr1a is found predominately in the extraembryonic and chorionic ectoderm from pregastrulation stages and is up-regulated within the embryo proper after gastrulation. Embryos with a germ line deletion of Pagr1a establish the anterior-posterior axis, and show normal neuroectodermal, mesodermal, and endodermal patterning, but fail to develop beyond the four- to five-somite stage or to undergo axial rotation. Pagr1a(-) (/) (-) embryos also show abnormal development of extraembryonic tissues with defects seen in the amnion, chorion and visceral yolk sac. At the molecular level, Pagr1a(-) (/) (-) embryos have reduced expression of BMP2, a known regulator of extraembryonic development., Conclusions: Loss of mouse Pagr1a function leads to defective extraembryonic development, likely due at least in part to altered BMP signaling, contributing to developmental arrest., (© 2014 Wiley Periodicals, Inc.)

Ion-exchange chromatography is the standard technique used for plasmid DNA purification, an essential molecular biology procedure. Non-ionic detergents (NIDs) have been used for plasmid DNA purification, but it is unclear whether Hofmeister series salts (HSS) change the solubility and phase separation properties of specific NIDs, enhancing plasmid DNA purification. After scaling-up NID-mediated plasmid DNA isolation, we established that NIDs in HSS solutions minimize plasmid DNA contamination with protein. In addition, large-scale NID/HSS solutions eliminated lipopolysaccharides (LPS) contamination of plasmid DNA more effectively than Qiagen ion-exchange columns. Large-scale NID isolation/NID purification generated increased yields of high-quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID-mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA-520 can be utilized for rapid, inexpensive, and efficient laboratory-based large-scale plasmid DNA purification, outperforming Qiagen-based column procedures., (Copyright © 2011 Wiley Periodicals, Inc.)

Nodal signaling is a critical regulator of multiple aspects of early vertebrate development including asymmetry along the left/right (LR) axis. To study Nodal function occurring specifically in the postgastrulation embryo, we have used Cre/loxP based conditional mutagenesis. A floxed allele of Nodal was generated and shown to have wild-type function. This allele was then used in conjunction with the T-Cre line, which expresses Cre recombinase broadly in the mesodermal and definitive endodermal lineages posterior to the cranial region. T-Cre activity leads to complete deletion of Nodal before its normal transient expression in the early somite stage lateral plate mesoderm, thereby causing severe LR developmental defects. No other abnormalities were found, suggesting that Nodal signaling has no additional essential functions in developmental patterning within the extensive mesodermal and endodermal domains marked by T-Cre activity.

A transgenic mouse strain derived from embryonic stem (ES) cells infected with multiple copies of a retroviral vector carries a recessive insertional mutation resulting in prenatal lethality. A detailed histological analysis of developing embryos has shown that the mutation results in hyperplasia of both embryonic and extraembryonic ectoderm and failure of mesoderm formation in the egg cylinder stage embryo. The number of cells in each lineage of normal and mutant embryos was estimated using stereological analysis of serial sections taken from implantation sites. We observed a 2-fold increase in the number of embryonic ectoderm cells in mutant embryos at 7.5 days postcoitum (dpc). In addition, we found that mutant embryonic ectoderm cells are only 0.6 times as large as normal cells. The number of extraembryonic ectoderm cells in mutant embryos at 7.5 dpc is also increased, by almost 4-fold. Mutant extraembryonic ectoderm cells are also smaller than normal, being only two-thirds the size of wild-type cells. The mutant phenotype suggests that the gene identified by this insertional mutation plays an important role in the growth control of early embryonic lineages.