Germ cell nuclear factor is not required for the down-regulation of pluripotency markers in fetal ovarian germ cells

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.Cataloged from PDF version of thesis.Includes bibliographical references.In mouse, germ cells retain expression of the pluripotency markers Oct4 and Nanog longer than any other cells in the body. While somatic cells repress these markers during gastrulation, female germ cells continue to express them until around the time of meiotic initiation. It is not yet clear why pluripotency markers are downregulated with this particular timing, nor is it understood what factors are involved in their repression. I have examined in fetal ovarian germ cells the expression and function of Gcnf (germ cell nuclear factor), an orphan nuclear receptor known to regulate both Oct4 and Nanog in gastrulating embryos. I have found that Gcnf is expressed in a female germ-cell-specific manner at the time when Oct4 and Nanog are down-regulated there. Gcnf mutants in which the ligand binding domain is disrupted display defects after gastrulation comparable to those observed in Gcnf-null mutants and those lacking the DNA binding domain. In contrast, the germ cells Gcnfligand binding domain mutants show no failure in repression of pluripotency markers, and other aspects of female germ cell development appear normal as well. Thus, it appears that the ligand binding domain of GCNF is not required for fetal ovarian germ cell development.by Leah M. Okumura.Ph.D

Parent Perception of Their Child’s Asthma Control and Concurrent Complementary and Alternative Medicine Use

We conducted a cross-sectional survey of 1322 parents of children with asthma to measure the prevalence and factors associated with complementary and alternative medicine (CAM) use for pediatric asthma. Multivariate regression techniques were used to determine factors associated with CAM use. Eleven percent (141/1322) of children were given CAM. Parents of children on daily medications who were perceived to have poor asthma control were almost three times more likely to use CAM than parents of children on no daily medications who were perceived to have high asthma control (risk ratio: = 2.81; confidence interval: 1.72, 4.60); age, gender, race, income, and education level were not significant independent predictors. Parent perception of asthma control is significantly associated with CAM use. It is important for providers to elicit information regarding CAM use in the clinic, as this may imply that the asthma symptoms may not be well controlled.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63130/1/pai.2008.0503.pd

The Ligand Binding Domain of GCNF Is Not Required for Repression of Pluripotency Genes in Mouse Fetal Ovarian Germ Cells

In mice, successful development and reproduction require that all cells, including germ cells, transition from a pluripotent to a differentiated state. This transition is associated with silencing of the pluripotency genes Oct4 and Nanog. Interestingly, these genes are repressed at different developmental timepoints in germ and somatic cells. Ovarian germ cells maintain their expression until about embryonic day (E) 14.5, whereas somatic cells silence them much earlier, at about E8.0. In both somatic cells and embryonic stem cells, silencing of Oct4 and Nanog requires the nuclear receptor GCNF. However, expression of the Gcnf gene has not been investigated in fetal ovarian germ cells, and whether it is required for silencing Oct4 and Nanog in that context is not known. Here we demonstrate that Gcnf is expressed in fetal ovarian germ cells, peaking at E14.5, when Oct4 and Nanog are silenced. However, conditional ablation of the ligand-binding domain of Gcnf using a ubiquitous, tamoxifen-inducible Cre indicates that Gcnf is not required for the down-regulation of pluripotency genes in fetal ovarian germ cells, nor is it required for initiation of meiosis and oogenesis. These results suggest that the silencing of Oct4 and Nanog in germ cells occurs via a different mechanism from that operating in somatic cells during gastrulation.Howard Hughes Medical InstituteNational Institutes of Health (U.S.) (2R01HG00257-20)National Human Genome Research Institute (U.S.) (2R01HG00257-20

Gata4 Is Required for Formation of the Genital Ridge in Mice

In mammals, both testis and ovary arise from a sexually undifferentiated precursor, the genital ridge, which first appears during mid-gestation as a thickening of the coelomic epithelium on the ventromedial surface of the mesonephros. At least four genes (Lhx9, Sf1, Wt1, and Emx2) have been demonstrated to be required for subsequent growth and maintenance of the genital ridge. However, no gene has been shown to be required for the initial thickening of the coelomic epithelium during genital ridge formation. We report that the transcription factor GATA4 is expressed in the coelomic epithelium of the genital ridge, progressing in an anterior-to-posterior (A-P) direction, immediately preceding an A-P wave of epithelial thickening. Mouse embryos conditionally deficient in Gata4 show no signs of gonadal initiation, as their coelomic epithelium remains a morphologically undifferentiated monolayer. The failure of genital ridge formation in Gata4-deficient embryos is corroborated by the absence of the early gonadal markers LHX9 and SF1. Our data indicate that GATA4 is required to initiate formation of the genital ridge in both XX and XY fetuses, prior to its previously reported role in testicular differentiation of the XY gonadHoward Hughes Medical Institut

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

<i>Gcnf</i> expression in fetal ovarian germ cells.

<p>(A) Levels of <i>Gcnf</i> transcript in female (XX) and male (XY) gonads from wild-type and germ-cell-depleted (<i>W/W^v</i>) mutant embryos at E12.5, E14.5, and E16.5, as determined by Illumina sequencing of gonadal RNA. Plotted here are average numbers of reads of <i>Gcnf</i> per million total reads from two individual biological replicates. (B) Single molecule fluorescence in situ hybridization for <i>Gcnf</i> mRNA (red) in sections of XX genital ridges or gonads, with germ cells marked by SSEA1 (green), and nuclei marked by DAPI staining (blue). Large red-orange spots in the E14.5 image are auto-fluorescent blood cells. Boxes indicate areas shown in higher magnification below each image. Scale bar, 20 um.</p

Migration of primordial germ cells is unaffected in <i>Gata4</i> cKO embryos.

<p>Whole-mount immunofluorescent staining for GATA4 protein (grey) in control and <i>Gata4</i> cKO (<i>Wt1^CreER</i>) embryos. Confocal images were taken sagittally for a longitudinal view of the genital ridge. <i>Oct4-EGFP</i> transgene marks germ cells (green). Panels on far right provide higher magnification views of boxed areas. Red dashed lines outline the coelomic epithelial surface. Scale bars: 50 µm.</p

<i>Gata4</i> deficiency impairs epithelial proliferation and basement membrane breakdown.

<p>(A) Immunofluorescent staining for BrdU (green) and laminin (red) in sections of control and <i>Gata4</i> cKO (<i>Wt1^CreER</i>;<i>Osr1^CreER</i>) embryos where tamoxifen and BrdU were injected at, respectively, E8.75 and ∼E10.0 (6 hours before sacrifice). Yellow dashed lines mark basement membrane. Yellow arrows mark discontinuities in basement membrane in control genital ridge. White arrows indicate representative BrdU-positive epithelial cells. Nuclei stained with DAPI (blue). ge, genital ridge epithelium; gm, genital mesenchyme. Scale bars: 50 µm. (B) Relative proliferation index, comparing the fractions of coelomic epithelial cells positive for BrdU in control and <i>Gata4</i> cKO (<i>Wt1^CreER</i>;<i>Osr1^CreER</i>) embryos of the same sex, from the same litter. The index in controls was set at 1. Germ cells were excluded from the counting. At each of the two stages shown, three pairs of control and <i>Gata4</i> cKO embryos were studied. Plotted here are means ± standard deviation. *, <i>P</i><0.05 (two-tailed Student's <i>t</i>-test).</p

<i>Gata4</i> is required for thickening of coelomic epithelium that gives rise to genital ridge.

<p>(A–E) IHC staining for GATA4 protein in transverse sections of control and <i>Gata4</i> cKO embryos. Sections were chosen to represent similar anterior positions in genital ridges. Right panel shows higher magnification of boxed area in left panel. Arrows indicate examples of positive GATA4 staining. Red dashed lines mark boundary between coelomic epithelium and underlying mesenchyme. Control and conditional mutant embryos, except for <i>Gata4</i> cKO (<i>CAG-CreER</i>), were from the same litter. a, dorsal aorta; g, gut endoderm; ge, genital ridge epithelium; gm, genital mesenchyme; m, mesentery; mt, mesonephric tubule. (F) Immunofluorescent staining for laminin protein in sections of control and <i>Gata4</i> cKO embryos. Laminin marks basement membrane. Control genital ridge shows thickened epithelial layer (yellow bracket), whereas the coelomic epithelium in <i>Rb</i> cKO embryo remains a single-cell layer (yellow dashed line). Scale bars: 50 µm.</p

GATA4 expression precedes coelomic epithelial thickening and progresses from anterior to posterior.

<p>(A) Schematic representation of experiment. Mouse embryos were dissected to remove limbs, body walls and internal organs, and then subjected to whole-mount immunofluorescence (IF) staining with GATA4 and SF1 antibodies. Stained embryos were imaged sagittally by confocal microscopy, and then transversely (following transverse section), again by confocal microscopy. Red dashed and solid lines in, respectively, sagittal and transverse images indicate location of developing gonads. a, dorsal aorta; m, mesentery; w, Wolffian duct. (B–D) Expression analysis of GATA4 (red) and SF1 (green) protein during early gonadogenesis. GATA4 expression in coelomic epithelia of genital ridges begins in anterior (arrow) and then spreads posteriorly. Epithelial thickening is observed in anterior region of genital ridge at 6-tail-somite stage (yellow arrowhead and inset). SF1 (white arrowheads) is expressed only sporadically in anterior half at 26–27 somite stage. Scale bars: 50 µm.</p