Design and fabrication of a chalcogenide hollow-core anti-resonant fiber for mid-infrared applications.

Chalcogenide hollow-core anti-resonant fibers (HC-ARFs) are a promising propagation medium for high-power mid-infrared (3-5 µm) laser delivery, while their properties have not been well understood and their fabrications remain challenging. In this paper, we design a seven-hole chalcogenide HC-ARF with touching cladding capillaries, which was then fabricated from purified As40S60 glass by combining the "stack-and-draw" method with a dual gas path pressure control technique. In particular, we predict theoretically and confirm experimentally that such medium exhibits higher-order mode suppression properties and several low-loss transmission bands in the mid-infrared spectrum, with the measured fiber loss being as low as 1.29 dB/m at 4.79 µm. Our results pave the way for the fabrication and implication of various chalcogenide HC-ARFs in mid-infrared laser delivery systems.

Timing and sequence requirements defined for embryonic maintenance of imprinted DNA methylation at Rasgrf1.

Epigenetic programming is critical for normal development of mammalian embryos. Errors cause misexpression of genes and aberrant development (E. Li, C. Beard, and R. Jaenisch, Nature 366:362-365, 1993). Imprinted genes are important targets of epigenetic regulation, but little is known about how the epigenetic patterns are established in the parental germ lines and maintained in the embryo. Paternal allele-specific expression at the imprinted Rasgrf1 locus in mice is controlled by paternal allele-specific methylation at a differentially methylated domain (DMD). DMD methylation is in turn controlled by a direct repeat sequence immediately downstream of the DMD which is required for establishing Rasgrf1 methylation in the male germ line (B. J. Yoon et al., Nat. Genet. 30:92-96, 2002). To determine if these repeats have a role in methylation maintenance, we developed a conditional deletion of the repeat sequence in mice and showed that the repeats are also required during a narrow interval to maintain paternal methylation of Rasgrf1 in developing embryos. Removing the repeats upon fertilization caused a total loss of methylation by the morula stage, but by the epiblast stage, the repeats were completely dispensable for methylation maintenance. This developmental interval coincides with genome-wide demethylation and remethylation in mice which most imprinted genes resist. Our data show that the Rasgrf1 repeats serve at least two functions: first, to establish Rasgrf1 DNA methylation in the male germ line, and second, to resist global demethylation in the preimplantation embryo.

Thoc1/Hpr1/p84 is essential for early embryonic development in the mouse.

The yeast TREX complex physically couples elongating RNA polymerase II with RNA processing and nuclear RNA export factors to facilitate regulated gene expression. Hpr1p is an essential component of TREX, and loss of Hpr1p compromises transcriptional elongation, RNA export, and genome stability. Despite these defects, HPR1 is not essential for viability in yeast. A functional orthologue of Hpr1p has been identified in metazoan species and is variously known as Thoc1, Hpr1, or p84. However, the physiological functions of this protein have not been determined. Here, we describe the generation and phenotypic characterization of mice containing a null allele of the Thoc1 gene. Heterozygous null Thoc1 mice are born at the expected Mendelian frequency with no phenotype distinguishable from the wild type. In contrast, homozygous null mice are not recovered, indicating that Thoc1 is required for embryonic development. Embryonic development is arrested around the time of implantation, as blastocysts exhibit hatching and blastocyst outgrowth defects upon in vitro culture. Cells of the inner cell mass are particularly dependent on Thoc1, as these cells rapidly lose viability coincident with Thoc1 protein loss. While Hpr1p is not essential for the viability of unicellular yeasts, the orthologous Thoc1 protein is required for viability of the early mouse embryo.

An E2F binding-deficient Rb1 protein partially rescues developmental defects associated with Rb1 nullizygosity.

Rb1 is essential for normal embryonic development, as null mice die in midgestation with widespread unscheduled cell proliferation. Rb1 protein (pRb) mediates cell cycle control by binding E2F transcription factors and repressing expression from E2F-dependent promoters. An increasing amount of evidence suggests that pRb loss also compromises cellular differentiation. Since differentiation is often dependent on cell cycle exit, it is currently unclear whether the effects of pRb on differentiation are an indirect consequence of pRb/E2F-mediated cell cycle control or whether they reflect direct cell-type-specific pRb functions. We have mutated Rb1 in the mouse to express a protein (R654W) specifically deficient in binding E2F1, E2F2, and E2F3. R654W mutant embryos exhibit cell cycle defects the same as those of Rb1 null embryos, reinforcing the importance of the interactions of pRb with E2F1, E2F2, and E2F3 for cell cycle control. However, R654W embryos survive at least 2 days longer than Rb1 null embryos, and increased life span is associated with improved erythrocyte and fetal liver macrophage differentiation. In contrast, R654W pRb does not rescue differentiation defects associated with pRb-deficient retinae. These data indicate that Rb1 makes important cell-type-specific contributions to cellular differentiation that are genetically separable from its general ability to stably bind E2F1, E2F2, and E2F3 and regulate the cell cycle.

Rasgrf1 imprinting is regulated by a CTCF-dependent methylation-sensitive enhancer blocker.

Imprinted methylation of the paternal Rasgrf1 allele in mice occurs at a differentially methylated domain (DMD) 30 kbp 5' of the promoter. A repeated sequence 3' of the DMD regulates imprinted methylation, which is required for imprinted expression. Here we identify the mechanism by which methylation controls imprinting. The DMD is an enhancer blocker that binds CTCF in a methylation-sensitive manner. CTCF bound to the unmethylated maternal allele silences expression. CTCF binding to the paternal allele is prevented by repeat-mediated methylation, allowing expression. Optimal in vitro enhancer-blocking activity requires CTCF binding sites. The enhancer blocker can be bypassed in vivo and imprinting abolished by placing an extra enhancer proximal to the promoter. Together, the repeats and the DMD constitute a binary switch that regulates Rasgrf1 imprinting.

Regulatory elements of the EKLF gene that direct erythroid cell-specific expression during mammalian development.

Erythroid Krüppel-like factor (EKLF) plays an essential role in enabling beta-globin expression during erythroid ontogeny. It is first expressed in the extraembryonic mesoderm of the yolk sac within the morphologically unique cells that give rise to the blood islands, and then later within the hepatic primordia. The BMP4/Smad pathway plays a critical role in the induction of EKLF, and transient transfection analyses demonstrate that sequences located within less than 1 kb of its transcription initiation site are sufficient for high-level erythroid-specific transcription. We have used transgenic analyses to verify that 950 bp located adjacent to the EKLF start site of transcription is sufficient to generate lacZ expression within the blood islands as well as the fetal liver during embryonic development. Of particular importance are 3 regions, 2 of which overlap endogenous erythroid-specific DNase hypersensitive sites, and 1 of which includes the proximal promoter region. The onset of transgene expression mimics that of endogenous EKLF as it begins by day 7.5 (d7.5) to d8.0. In addition, it exhibits a strict hematopoietic specificity, localized only to these cells and not to the adjacent vasculature at all stages examined. Finally, expression is heterocellular, implying that although these elements are sufficient for tissue-specific expression, they do not shield against the position effects of adjacent chromatin. These analyses demonstrate that a surprisingly small DNA segment contains all the information needed to target a linked gene to the hematopoietic compartment at both early and later stages of development, and may be a useful cassette for this purpose.

Trans allele methylation and paramutation-like effects in mice.

In mammals, imprinted genes have parent-of-origin-specific patterns of DNA methylation that cause allele-specific expression. At Rasgrf1 (encoding RAS protein-specific guanine nucleotide-releasing factor 1), a repeated DNA element is needed to establish methylation and expression of the active paternal allele. At Igf2r (encoding insulin-like growth factor 2 receptor), a sequence called region 2 is needed for methylation of the active maternal allele. Here we show that replacing the Rasgrf1 repeats on the paternal allele with region 2 allows both methylation and expression of the paternal copy of Rasgrf1, indicating that sequences that control methylation can function ectopically. Paternal transmission of the mutated allele also induced methylation and expression in trans of the normally unmethylated and silent wild-type maternal allele. Once activated, the wild-type maternal Rasgrf1 allele maintained its activated state in the next generation independently of the paternal allele. These results recapitulate in mice several features in common with paramutation described in plants.