PSF1 is essential for early embryogenesis in mice

Psf1 (partner of sld five 1) forms a novel heterotetramer complex, GINS (Go, Ichi, Nii, and San; five, one, two, and three, respectively, in Japanese), with Sld5, Psf2, and Psf3. The formation of this complex is essential for the initiation of DNA replication in yeast and Xenopus laevis egg extracts. Although all of the components are well conserved in higher eukaryotes, the biological function in vivo is largely unknown. We originally cloned the mouse ortholog of PSF1 from a hematopoietic stem cell cDNA library and found that PSF1 is expressed in blastocysts, adult bone marrow, and testis, in which the stem cell system is active. Here we used the gene-targeting technique to determine the physiological function of PSF1 in vivo. Mice homozygous for a nonfunctional mutant of PSF1 died in utero around the time of implantation. PSF1-/- blastocysts failed to show outgrowth in culture and exhibited a cell proliferation defect. Our data clearly indicate that PSF1 is required for early embryogenesis.     

Arabidopsis EED1 encoding a plant-specific nuclear protein is essential for early embryogenesis

正Embryogenesis is central to the life cycle of higher plants, which starts from division of the single-cell zygote and culminates in formation of the mature embryo with major cell precursors. Such a process generally involves tightly orchestrated cell division, differentiation, and pattern formation, which is precisely controlled by a complicated gene regulatory network(Jenik et al., 2007; Capron et al., 2009; Wendrich and Weijers, 2013; ten Hove et al., 2015;     

Targeting an Essential GTPase Obg for the Development of Broad-Spectrum Antibiotics

A promising new drug target for the development of novel broad-spectrum antibiotics is the highly conserved small GTPase Obg (YhbZ, CgtA), a protein essential for the survival of all bacteria including Neisseria gonorrhoeae (GC). GC is the agent of gonorrhea, a prevalent sexually transmitted disease resulting in serious consequences on reproductive and neonatal health. A preventive anti-gonorrhea vaccine does not exist, and options for effective antibiotic treatments are increasingly limited. To address the dire need for alternative antimicrobial strategies, we have designed and optimized a 384-well GTPase assay to identify inhibitors of Obg using as a model Obg protein from GC, Obg_GC. The assay was validated with a pilot screen of 40,000 compounds and achieved an average Z’ value of 0.58 ± 0.02, which suggests a robust assay amenable to high-throughput screening. We developed secondary assessments for identified lead compounds that utilize the interaction between Obg_GC and fluorescent guanine nucleotide analogs, mant-GTP and mant-GDP, and an Obg_GC variant with multiple alterations in the G-domains that prevent nucleotide binding. To evaluate the broad-spectrum potential of Obg_GC inhibitors, Obg proteins of Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus were assessed using the colorimetric and fluorescence-based activity assays. These approaches can be useful in identifying broad-spectrum Obg inhibitors and advancing the therapeutic battle against multidrug resistant bacteria.     

SEBOX is essential for early embryogenesis at the two-cell stage in the mouse

Previously, we found high levels of skin-embryo-brain-oocyte homeobox (Sebox) gene expression in germinal vesicle (GV)-stage oocytes. The objective of the present study was to determine the role played by SEBOX in oocyte maturation and early embryogenesis using RNA interference (RNAi). Microinjection of Sebox double-stranded RNA into GV oocytes resulted in a marked decrease in Sebox mRNA and protein expression. However, Sebox RNAi affects neither oocyte maturation rate nor morphological characteristics, including spindle and chromosomal organization of metaphase II oocytes. In addition, Sebox RNAi had no discernible effect on the activities of M-phase promoting factor or mitogen-activated protein kinase. In contrast, microinjection of Sebox double-stranded RNA into pronuclear-stage embryos resulted in holding embryo development at the two-cell (84.9%) and the four- and eight-cell (15.1%) stages. We concluded that Sebox is a new addition to maternal effect genes that produced and stored in oocytes and function in preimplantation embryo development.     

Activity-regulated, cytoskeleton-associated protein (Arc) is essential for visceral endoderm organization during early embryogenesis

Activity-regulated, cytoskeleton-associated protein (Arc) was first identified as an immediate-early gene regulated by synaptic activity. We have studied its functional role in vivo using a gene-targeting approach. We found that Arc is encoded by a single exon, and Arc mRNA is ubiquitously expressed in early mouse embryos. Homozygous Arc mutants are severely growth-retarded, fail to gastrulate and subsequently die before day 8.5 of embryogenesis. Further analysis revealed severe disorganization of visceral endoderm formation, and total separation and ectopic location of embryonic and extraembryonic structure. These findings demonstrate that Arc function is essential for early embryo development and patterning in mice, and support the hypothesis that signaling from visceral endoderm is essential for normal patterning of the extraembryonic and embryonic structure.     

Elf5 is essential for early embryogenesis and mammary gland development during pregnancy and lactation

Elf5 is an epithelial-specific ETS factor. Embryos with a null mutation in the Elf5 gene died before embryonic day 7.5, indicating that Elf5 is essential during mouse embryogenesis. Elf5 is also required for proliferation and differentiation of mouse mammary alveolar epithelial cells during pregnancy and lactation. The loss of one functional allele led to complete developmental arrest of the mammary gland in pregnant Elf5 heterozygous mice. A quantitative mRNA expression study and Western blot analysis revealed that decreased expression of Elf5 correlated with the downregulation of milk proteins in Elf5(+/-) mammary glands. Mammary gland transplants into Rag(-/-) mice demonstrated that Elf5(+/-) mammary alveolar buds failed to develop in an Elf5(+/+) mammary fat pad during pregnancy, demonstrating an epithelial cell autonomous defect. Elf5 expression was reduced in Prolactin receptor (Prlr) heterozygous mammary glands, which phenocopy Elf5(+/-) glands, suggesting that Elf5 and Prlr are in the same pathway. Our data demonstrate that Elf5 is essential for developmental processes in the embryo and in the mammary gland during pregnancy.     

Mgm1p,a dynamin-related GTPase,is essential for fusion of the mitochondrial outer membrane

In Saccharomyces cerevisiae, mitochondrial fusion requires at least two outer membrane proteins, Fzo1p and Ugo1p. We provide direct evidence that the dynamin-related Mgm1 protein is also required for mitochondrial fusion. Like fzo1 and ugo1 mutants, cells disrupted for the MGM1 gene contain numerous mitochondrial fragments instead of the few long, tubular organelles seen in wild-type cells. Fragmentation of mitochondria in mgm1 mutants is rescued by disrupting DNM1, a gene required for mitochondrial division. In zygotes formed by mating mgm1 mutants, mitochondria do not fuse and mix their contents. Introducing mutations in the GTPase domain of Mgm1p completely block mitochondrial fusion. Furthermore, we show that mgm1 mutants fail to fuse both their mitochondrial outer and inner membranes. Electron microscopy demonstrates that although mgm1 mutants display aberrant mitochondrial inner membrane cristae, mgm1 dnm1 double mutants restore normal inner membrane structures. However, mgm1 dnm1 mutants remain defective in mitochondrial fusion, indicating that mitochondrial fusion requires Mgm1p regardless of the morphology of mitochondria. Finally, we find that Mgm1p, Fzo1p, and Ugo1p physically interact in the mitochondrial outer membrane. Our results raise the possibility that Mgm1p regulates fusion of the mitochondrial outer membrane through its interactions with Fzo1p and Ugo1p.     

CDC-25.2, a C. elegans ortholog of cdc25, is essential for the progression of intestinal divisions

Intestinal divisions in Caenorhabditis elegans take place in 3 stages: (1) cell divisions during embryogenesis, (2) binucleations at the L1 stage, and (3) endoreduplications at the end of each larval stage. Here, we report that CDC-25.2, a C. elegans ortholog of Cdc25, is required for these specialized division cycles between the 16E cell stage and the onset of endoreduplication. Results of our genetic analyses suggest that CDC-25.2 regulates intestinal cell divisions and binucleations by counteracting WEE-1.3 and by activating the CDK-1/CYB-1 complex. CDC-25.2 activity is then repressed by LIN-23 E3 ubiquitin ligase before the onset of intestinal endoreduplication, and this repression is maintained by LIN-35, the C. elegans ortholog of Retinoblastoma (Rb). These findings indicate that timely regulation of CDC-25.2 activity is essential for the progression of specialized division cycles and development of the C. elegans intestine.