Epigenetic reprogramming in the porcine germ line

<p>Abstract</p> <p>Background</p> <p>Epigenetic reprogramming is critical for genome regulation during germ line development. Genome-wide demethylation in mouse primordial germ cells (PGC) is a unique reprogramming event essential for erasing epigenetic memory and preventing the transmission of epimutations to the next generation. In addition to DNA demethylation, PGC are subject to a major reprogramming of histone marks, and many of these changes are concurrent with a cell cycle arrest in the G2 phase. There is limited information on how well conserved these events are in mammals. Here we report on the dynamic reprogramming of DNA methylation at CpGs of imprinted loci and DNA repeats, and the global changes in H3K27me3 and H3K9me2 in the developing germ line of the domestic pig.</p> <p>Results</p> <p>Our results show loss of DNA methylation in PGC colonizing the genital ridges. Analysis of <it>IGF2-H19 </it>regulatory region showed a gradual demethylation between E22-E42. In contrast, DMR2 of <it>IGF2R </it>was already demethylated in male PGC by E22. In females, <it>IGF2R </it>demethylation was delayed until E29-31, and was de novo methylated by E42. DNA repeats were gradually demethylated from E25 to E29-31, and became de novo methylated by E42. Analysis of histone marks showed strong H3K27me3 staining in migratory PGC between E15 and E21. In contrast, H3K9me2 signal was low in PGC by E15 and completely erased by E21. Cell cycle analysis of gonadal PGC (E22-31) showed a typical pattern of cycling cells, however, migrating PGC (E17) showed an increased proportion of cells in G2.</p> <p>Conclusions</p> <p>Our study demonstrates that epigenetic reprogramming occurs in pig migratory and gonadal PGC, and establishes the window of time for the occurrence of these events. Reprogramming of histone H3K9me2 and H3K27me3 detected between E15-E21 precedes the dynamic DNA demethylation at imprinted loci and DNA repeats between E22-E42. Our findings demonstrate that major epigenetic reprogramming in the pig germ line follows the overall dynamics shown in mice, suggesting that epigenetic reprogramming of germ cells is conserved in mammals. A better understanding of the sequential reprogramming of PGC in the pig will facilitate the derivation of embryonic germ cells in this species.</p

In vitro culture of embryonic mouse intestinal epithelium: cell differentiation and introduction of reporter genes

BACKGROUND: Study of the normal development of the intestinal epithelium has been hampered by a lack of suitable model systems, in particular ones that enable the introduction of exogenous genes. Production of such a system would advance our understanding of normal epithelial development and help to shed light on the pathogenesis of intestinal neoplasia. The criteria for a reliable culture system include the ability to perform real time observations and manipulations in vitro, the preparation of wholemounts for immunostaining and the potential for introducing genes. RESULTS: The new culture system involves growing mouse embryo intestinal explants on fibronectin-coated coverslips in basal Eagle's medium+20% fetal bovine serum. Initially the cultures maintain expression of the intestinal transcription factor Cdx2 together with columnar epithelial (cytokeratin 8) and mesenchymal (smooth muscle actin) markers. Over a few days of culture, differentiation markers appear characteristic of absorptive epithelium (sucrase-isomaltase), goblet cells (Periodic Acid Schiff positive), enteroendocrine cells (chromogranin A) and Paneth cells (lysozyme). Three different approaches were tested to express genes in the developing cultures: transfection, electroporation and adenoviral infection. All could introduce genes into the mesenchyme, but only to a small extent into the epithelium. However the efficiency of adenovirus infection can be greatly improved by a limited enzyme digestion, which makes accessible the lateral faces of cells bearing the Coxsackie and Adenovirus Receptor. This enables reliable delivery of genes into epithelial cells. CONCLUSION: We describe a new in vitro culture system for the small intestine of the mouse embryo that recapitulates its normal development. The system both provides a model for studying normal development of the intestinal epithelium and also allows for the manipulation of gene expression. The explants can be cultured for up to two weeks, they form the full repertoire of intestinal epithelial cell types (enterocytes, goblet cells, Paneth cells and enteroendocrine cells) and the method for gene introduction into the epithelium is efficient and reliable

Pseudomonas aeruginosa Enhances Production of a Non-Alginate Exopolysaccharide during Long-Term Colonization of the Cystic Fibrosis Lung

The gram-negative opportunistic pathogen Pseudomonas aeruginosa is the primary cause of chronic respiratory infections in individuals with the heritable disease cystic fibrosis (CF). These infections can last for decades, during which time P. aeruginosa has been proposed to acquire beneficial traits via adaptive evolution. Because CF lacks an animal model that can acquire chronic P. aeruginosa infections, identifying genes important for long-term in vivo fitness remains difficult. However, since clonal, chronological samples can be obtained from chronically infected individuals, traits undergoing adaptive evolution can be identified. Recently we identified 24 P. aeruginosa gene expression traits undergoing parallel evolution in vivo in multiple individuals, suggesting they are beneficial to the bacterium. The goal of this study was to determine if these genes impact P. aeruginosa phenotypes important for survival in the CF lung. By using a gain-of-function genetic screen, we found that 4 genes and 2 operons undergoing parallel evolution in vivo promote P. aeruginosa biofilm formation. These genes/operons promote biofilm formation by increasing levels of the non-alginate exopolysaccharide Psl. One of these genes, phaF, enhances Psl production via a post-transcriptional mechanism, while the other 5 genes/operons do not act on either psl transcription or translation. Together, these data demonstrate that P. aeruginosa has evolved at least two pathways to over-produce a non-alginate exopolysaccharide during long-term colonization of the CF lung. More broadly, this approach allowed us to attribute a biological significance to genes with unknown function, demonstrating the power of using evolution as a guide for targeted genetic studies.open6

Constraints on the thermal evolution of metamorphic core complexes from the timing of high-pressure metamorphism on Naxos, Greece

Metamorphic core complexes are classically interpreted to have formed during crustal extension, although many also occur in compressional environments. New U–(Th)–Pb allanite and xenotime geochronologic data from the structurally highest Zas Unit (Cycladic Blueschist Unit) of the Naxos metamorphic core complex, Greece, integrated with pressure–temperature–time (P–T–t) histories, are incorporated into a thermal model to test the role of crustal thickening and extension in forming metamorphic core complexes. Metamorphism on Naxos is diachronous, with peak metamorphic conditions propagating down structural section over a ~30–35 m.y. period, from ca. 50 Ma to 15 Ma. At the highest structural level, the Zas Unit records blueschist-facies metamorphism (~14.5–19 kbar, 470–570 °C) at ca. 50 Ma, during northeast-directed subduction of the Adriatic continental margin. The Zas Unit was subsequently extruded toward the SW and thrust over more proximal continental margin and basement rocks (Koronos and Core units). This contractional episode resulted in crustal thickening and Barrovian metamorphism from ca. 40 Ma and reached peak kyanite-sillimanite–grade conditions of ~10–5 kbar and 600–730 °C at 20–15 Ma. Model P–T–t paths, assuming conductive relaxation of isotherms following overthrusting, are consistent with the clockwise P–T–t evolution. In contrast, extension results in exhumation and cooling of the crust, which is inconsistent with key components of the thermal evolution. Barrovian metamorphism on Naxos is therefore interpreted to have resulted from crustal thickening over a ~30–35 m.y. time period prior to extension, normal faulting, and rapid exhumation after a thermal climax at ca. 15 Ma