The Influence of Polyploidy and Genome Composition on Genomic Imprinting in Mice

Genomic imprinting is an epigenetic mechanism that switches the expression of imprinted genes involved in normal embryonic growth and development in a parent-of-origin-specific manner. Changes inDNAmethylation statuses from polyploidization are a well characterized epigenetic modification in plants. However, how changes in ploidy affect both imprinted gene expression and methylation status in mammals remains unclear. To address this, we used quantitative real time PCR to analyze expression levels of imprinted genes in mouse tetraploid fetuses. We used bisulfite sequencing to assess the methylation statuses of differentially methylated regions (DMRs) that regulate imprinted gene expression in triploid and tetraploid fetuses. The nine imprinted genes H19, Gtl2, Dlk1, Igf2r, Grb10, Zim1, Peg3, Ndn, and Ipw were all unregulated; in particular, the expression of Zim1 was more than 10-fold higher, and the expression of Ipw was repressed in tetraploid fetuses. The methylation statuses of four DMRs H19, intergenic (IG), Igf2r, and Snrpn in tetraploid and triploid fetuses were similar to those in diploid fetuses. We also performed allele-specific RT-PCR sequencing to determine the alleles expressing the three imprinted genes Igf2, Gtl2, and Dlk1 in tetraploid fetuses. These three imprinted genes showed monoallelic expression in a parent-of-origin-specific manner. Expression of non-imprinted genes regulating neural cell development significantly decreased in tetraploid fetuses, which might have been associated with unregulated imprinted gene expression. This study provides the first detailed analysis of genomic imprinting in tetraploid fetuses, suggesting that imprinted gene expression is disrupted, but DNA methylation statuses of DMRs are stable following changes in ploidy in mammals

Chromosomal Dynamics at the Shh Locus: Limb Bud-Specific Differential Regulation of Competence and Active Transcription

The expression of Sonic hedgehog (Shh) in mouse limb buds is regulated by a long-range enhancer 1 Mb upstream of the Shh promoter. We used 3D-FISH and chromosome conformation capture assays to track changes at the Shh locus and found that long-range promoter-enhancer interactions are specific to limb bud tissues competent to express Shh. However, the Shh locus loops out from its chromosome territory only in the posterior limb bud (zone of polarizing activity or ZPA), where Shh expression is active. Notably, while Shh mRNA is detected throughout the ZPA, enhancer-promoter interactions and looping out were only observed in small fractions of ZPA cells. In situ detection of nascent Shh transcripts and unstable EGFP reporters revealed that active Shh transcription is likewise only seen in a small fraction of ZPA cells. These results suggest that chromosome conformation dynamics at the Shh locus allow transient pulses of Shh transcription

The Role of Echocardiography in the Management of Patients Undergoing a Ventricular Assist Device Implantation and/or Transplantation

Heart transplantation (HTx) is a curative treatment for patients with advanced heart failure (HF); however, since transplant opportunities are severely limited due to donor shortage, the left ventricular assist device (LVAD) has become a standard therapy for patients awaiting HTx. The role of echocardiography as a primary imaging modality to monitor the allograft function in transplant recipients as well as to optimize LVAD settings in LVAD recipients has been expanding. The purpose of this review is to highlight the clinical role of echocardiography in the management of patients undergoing LVAD implantation and/or HTx. In particular, we overview (1) how to detect LVAD malfunction and device-associated complication in LVAD recipients and (2) echocardiographic assessments of cardiac allograft rejection in transplant recipients

Chromosomal Dynamics at the Shh Locus: Limb Bud-Specific Differential Regulation of Competence and Active Transcription

SummaryThe expression of Sonic hedgehog (Shh) in mouse limb buds is regulated by a long-range enhancer 1 Mb upstream of the Shh promoter. We used 3D-FISH and chromosome conformation capture assays to track changes at the Shh locus and found that long-range promoter-enhancer interactions are specific to limb bud tissues competent to express Shh. However, the Shh locus loops out from its chromosome territory only in the posterior limb bud (zone of polarizing activity or ZPA), where Shh expression is active. Notably, while Shh mRNA is detected throughout the ZPA, enhancer-promoter interactions and looping out were only observed in small fractions of ZPA cells. In situ detection of nascent Shh transcripts and unstable EGFP reporters revealed that active Shh transcription is likewise only seen in a small fraction of ZPA cells. These results suggest that chromosome conformation dynamics at the Shh locus allow transient pulses of Shh transcription

Identification and targeted disruption of the mouse gene encoding ESG1 (PH34/ECAT2/DPPA5)

BACKGROUND: Embryonic stem cell-specific gene (ESG) 1, which encodes a KH-domain containing protein, is specifically expressed in early embryos, germ cells, and embryonic stem (ES) cells. Previous studies identified genomic clones containing the mouse ESG1 gene and five pseudogenes. However, their chromosomal localizations or physiological functions have not been determined. RESULTS: A Blast search of mouse genomic databases failed to locate the ESG1 gene. We identified several bacterial artificial clones containing the mouse ESG1 gene and an additional ESG1-like sequence with a similar gene structure from chromosome 9. The ESG1-like sequence contained a multiple critical mutations, indicating that it was a duplicated pseudogene. The 5' flanking region of the ESG1 gene, but not that of the pseudogene, exhibited strong enhancer and promoter activity in undifferentiated ES cells by luciferase reporter assay. To study the physiological functions of the ESG1 gene, we replaced this sequence in ES cells with a β-geo cassette by homologous recombination. Despite specific expression in early embryos and germ cells, ESG1(-/- )mice developed normally and were fertile. We also generated ESG1(-/- )ES cells both by a second independent homologous recombination and directly from blastocysts derived from heterozygous intercrosses. Northern blot and western blot analyses confirmed the absence of ESG1 in these cells. These ES cells demonstrated normal morphology, proliferation, and differentiation. CONCLUSION: The mouse ESG1 gene, together with a duplicated pseudogene, is located on chromosome 9. Despite its specific expression in pluripotent cells and germ cells, ESG1 is dispensable for self-renewal of ES cells and establishment of germcells

A Crucial Role of Bone Morphogenetic Protein Signaling in the Wound Healing Response in Acute Liver Injury Induced by Carbon Tetrachloride

Background. Acute liver injury induced by administration of carbon tetrachloride (CCl4) has used a model of wound repair in the rat liver. Previously, we reported transient expression of bone morphogenetic protein (Bmp) 2 or Bmp4 at 6–24 h after CCl4 treatment, suggesting a role of BMP signaling in the wound healing response in the injured liver. In the present study, we investigated the biological meaning of the transient Bmp expression in liver injury. Methods. Using conditional knockout mice carrying a floxed exon in the BMP receptor 1A gene, we determined the hepatic gene expressions and proliferative activity following CCl4-treated liver. Results. We observed retardation of the healing response in the knockout mice treated with CCl4, including aggravated histological feature and reduced expressions of the albumin and Tdo2 genes, and a particular decrease in the proliferative activity shown by Ki-67 immunohistochemistry. Conclusion. Our findings suggest a crucial role of BMP signaling in the amelioration of acute liver injury

Normalization of Pulmonary Hypertension by the Use of Left Ventricular Assist Device in Patients with End-stage Heart Failure: A Possible Contribution to Donor Pool Expansion in Lung Transplantation

SummaryHeart transplantation alone has been recognized to be contraindicated when pulmonary hypertension (PH) and elevated pulmonary vascular resistance (PVR) are irreversible, irrespective of any medical intervention by the use of inotropic agents or pulmonary vasodilators, because such patients are at an increased risk of post-transplantation right ventricular failure and mortality. Therefore, end-stage heart failure patients with concomitant fixed PH and irreversibly high PVR are considered to be heart–lung transplant candidates. Recently, left ventricular assist device (LVAD) therapy has been reported to normalize PVR through persistent unloading of the left ventricle, even in patients with medically refractory PH. Therefore, LVAD therapy could make such patients suitable for “heart-only” transplants, which contributes to appropriate donor lung allocation for lung-only candidates. We review the literature regarding LVAD use for secondary PH and present a case with end-stage heart failure that could avoid a heart–lung transplant owing to LVAD therapy

Localization of Heat Shock Protein 27 (Hsp27) in the Rat Gingiva and its Changes with Tooth Eruption

Heat shock protein 27 kDa (Hsp27) functions as a molecular chaperon to prevent apoptosis as well as to contribute to the regulation of cell proliferation and differentiation during development. In the present study, the localization of Hsp27 in the oral epithelium of rats and its expression change during formation of the gingiva with the tooth eruption were examined immunohistochemically to elucidate the roles of Hsp27 in the oral mucosa