scholarly journals The Nuclear Scaffold Protein NIPP1 Is Essential for Early Embryonic Development and Cell Proliferation

2004 ◽  
Vol 24 (13) ◽  
pp. 5863-5874 ◽  
Author(s):  
Aleyde Van Eynde ◽  
Mieke Nuytten ◽  
Mieke Dewerchin ◽  
Luc Schoonjans ◽  
Stefaan Keppens ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Embryonic Development ◽  
Protein Phosphatase ◽  
Cultured Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Scaffold Protein ◽  
Early Embryonic Development ◽  
Nuclear Scaffold

ABSTRACT NIPP1 (nuclear inhibitor of protein phosphatase 1) is a ubiquitously expressed nuclear scaffold protein that has been implicated in both transcription and RNA processing. Among its protein ligands are a protein kinase, a protein phosphatase, two splicing factors, and a transcriptional regulator, and the binding of these proteins to NIPP1 is tightly regulated by phosphorylation. To study the function of NIPP1 in vivo, we have used homologous recombination to generate mice that are deficient in NIPP1. NIPP1−/+ mice developed normally. However, NIPP1−/− embryos showed severely retarded growth at embryonic day 6.5 (E6.5) and were resorbed by E8.5. This early embryonic lethality was not associated with increased apoptosis but correlated with impaired cell proliferation. Blastocyst outgrowth experiments and the RNA interference-mediated knockdown of NIPP1 in cultured cells also revealed an essential role for NIPP1 in cell proliferation. In further agreement with this function, no viable NIPP1−/− cell lines were obtained by derivation of embryonic stem (ES) cells from blastocysts of NIPP1−/+ intercrosses or by forced homogenotization of heterozygous ES cells at high concentrations of Geneticin. We conclude that NIPP1 is indispensable for early embryonic development and cell proliferation.

scholarly journals Murine Surf4 is essential for early embryonic development

2019 ◽  
Author(s):  
Brian T. Emmer ◽  
Paul J. Lascuna ◽  
Emilee N. Kotnik ◽  
Thomas L. Saunders ◽  
Rami Khoriaty ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Apolipoprotein B ◽  
Gene Editing ◽  
Cultured Cells ◽  
Early Embryonic Development ◽  
Loss Of Function ◽  
Normal Appearance ◽  
Anterograde Transport ◽  
Transport Vesicles

ABSTRACTNewly synthesized proteins co-translationally inserted into the endoplasmic reticulum (ER) lumen may be recruited into anterograde transport vesicles by their association with specific cargo receptors. We recently identified a role for the cargo receptor SURF4 in facilitating the secretion of PCSK9 in cultured cells. To examine the function of SURF4 in vivo, we used CRISPR/Cas9-mediated gene editing to generate mice with germline loss-of-function mutations in Surf4. Surf4+/- mice exhibited grossly normal appearance, behavior, body weight, fecundity, and organ development and demonstrated no significant alterations in circulating plasma levels of PCSK9, apolipoprotein B, or total cholesterol. Surf4-/- mice exhibit embryonic lethality, with complete loss of all Surf4-/- offspring between embryonic days 3.5 and 9.5. Taken together with the much milder phenotypes of PCSK9 or apolipoprotein B deficiency in mice, these findings imply the existence of additional SURF4 cargoes or functions that are essential for murine early embryonic development.

scholarly journals Human Hematopoietic Stem Cells Can Survive In Vitro for Several Months

2009 ◽  
Vol 2009 ◽  
pp. 1-7 ◽  
Author(s):  
Taro Ishigaki ◽  
Kazuhiro Sudo ◽  
Takashi Hiroyama ◽  
Kenichi Miharada ◽  
Haruhiko Ninomiya ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cultured Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Cell Sample

We previously reported that long-lasting in vitro hematopoiesis could be achieved using the cells differentiated from primate embryonic stem (ES) cells. Thus, we speculated that hematopoietic stem cells differentiated from ES cells could sustain long-lasting in vitro hematopoiesis. To test this hypothesis, we investigated whether human hematopoietic stem cells could similarly sustain long-lasting in vitro hematopoiesis in the same culture system. Although the results varied between experiments, presumably due to differences in the quality of each hematopoietic stem cell sample, long-lasting in vitro hematopoiesis was observed to last up to nine months. Furthermore, an in vivo analysis in which cultured cells were transplanted into immunodeficient mice indicated that even after several months of culture, hematopoietic stem cells were still present in the cultured cells. To the best of our knowledge, this is the first report to show that human hematopoietic stem cells can survive in vitro for several months.

scholarly journals Dp1 Is Largely Dispensable for Embryonic Development

2004 ◽  
Vol 24 (16) ◽  
pp. 7197-7205 ◽  
Author(s):  
Matthew J. Kohn ◽  
Sandra W. Leung ◽  
Vittoria Criniti ◽  
Monica Agromayor ◽  
Lili Yamasaki
Keyword(s):  
Cell Cycle ◽  
Embryonic Development ◽  
Cell Cycle Progression ◽  
Target Genes ◽  
Es Cells ◽  
Embryonic Stem ◽  
Wild Type ◽  
Cycle Progression ◽  
Cell Cycle Genes

ABSTRACT E2F/DP complexes activate or repress the transcription of E2F target genes, depending on the association of a pRB family member, thereby regulating cell cycle progression. Whereas the E2F family consists of seven members, the DP family contains only two (Dp1 and Dp2), Dp1 being the more highly expressed member. In contrast to the inactivation of individual E2F family members, we have recently demonstrated that loss of Dp1 results in embryonic lethality by embryonic day 12.5 (E12.5) due to the failure of extraembryonic lineages to develop and replicate DNA properly. To bypass this placental requirement and search for roles of Dp1 in the embryo proper, we generated Dp1-deficient embryonic stem (ES) cells that carry the ROSA26-LacZ marker and injected them into wild-type blastocysts to construct Dp1-deficient chimeras. Surprisingly, we recovered mid- to late gestational embryos (E12.5 to E17.5), in which the Dp1-deficient ES cells contributed strongly to most chimeric tissues as judged by X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) staining and Western blotting. Importantly, the abundance of DP2 protein does not increase and the expression of an array of cell cycle genes is virtually unchanged in Dp1-deficient ES cells or chimeric E15.5 tissues with the absence of Dp1. Thus, Dp1 is largely dispensable for embryonic development, despite the absolute extraembryonic requirement for Dp1, which is highly reminiscent of the restricted roles for Rb and cyclins E1/E2 in vivo.

scholarly journals The roles of ERAS during cell lineage specification of mouse early embryonic development

Open Biology ◽  
2015 ◽  
Vol 5 (8) ◽  
pp. 150092 ◽  
Author(s):  
Zhen-Ao Zhao ◽  
Yang Yu ◽  
Huai-Xiao Ma ◽  
Xiao-Xiao Wang ◽  
Xukun Lu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
Embryonic Stem Cells ◽  
Embryonic Development ◽  
Expression Pattern ◽  
Embryonic Stem ◽  
Cell Lineage ◽  
Primitive Streak ◽  
Early Embryonic Development ◽  
Lineage Specification

Eras encodes a Ras-like GTPase protein that was originally identified as an embryonic stem cell-specific Ras. ERAS has been known to be required for the growth of embryonic stem cells and stimulates somatic cell reprogramming, suggesting its roles on mouse early embryonic development. We now report a dynamic expression pattern of Eras during mouse peri-implantation development: its expression increases at the blastocyst stage, and specifically decreases in E7.5 mesoderm. In accordance with its expression pattern, the increased expression of Eras promotes cell proliferation through controlling AKT activation and the commitment from ground to primed state through ERK activation in mouse embryonic stem cells; and the reduced expression of Eras facilitates primitive streak and mesoderm formation through AKT inhibition during gastrulation. The expression of Eras is finely regulated to match its roles in mouse early embryonic development during which Eras expression is negatively regulated by the β -catenin pathway. Thus, beyond its well-known role on cell proliferation, ERAS may also play important roles in cell lineage specification during mouse early embryonic development.

scholarly journals Essential Role of AKT-1/Protein Kinase Bα in PTEN-Controlled Tumorigenesis

2002 ◽  
Vol 22 (11) ◽  
pp. 3842-3851 ◽  
Author(s):  
Bangyan Stiles ◽  
Valeriya Gilman ◽  
Natalya Khanzenzon ◽  
Ralf Lesche ◽  
Annie Li ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Survival ◽  
Essential Role ◽  
Es Cells ◽  
Familial Cancer ◽  
Embryonic Stem ◽  
Cellular Processes ◽  
Downstream Effectors

ABSTRACT PTEN is mutated at high frequency in many primary human cancers and several familial cancer predisposition disorders. Activation of AKT is a common event in tumors in which the PTEN gene has been inactivated. We previously showed that deletion of the murine Pten gene in embryonic stem (ES) cells led to increased phosphatidylinositol triphosphate (PIP3) accumulation, enhanced entry into S phase, and better cell survival. Since PIP3 controls multiple signaling molecules, it was not clear to what degree the observed phenotypes were due to deregulated AKT activity. In this study, we mutated Akt-1 in Pten −/− ES cells to directly assess the role of AKT-1 in PTEN-controlled cellular processes, such as cell proliferation, cell survival, and tumorigenesis in nude mice. We showed that AKT-1 is one of the major downstream effectors of PTEN in ES cells and that activation of AKT-1 is required for both the cell survival and cell proliferation phenotypes observed in Pten −/− ES cells. Deletion of Akt-1 partially reverses the aggressive growth of Pten −/− ES cells in vivo, suggesting that AKT-1 plays an essential role in PTEN-controlled tumorigenesis.

scholarly journals MRG15 Regulates Embryonic Development and Cell Proliferation

2005 ◽  
Vol 25 (8) ◽  
pp. 2924-2937 ◽  
Author(s):  
Kaoru Tominaga ◽  
Bhakti Kirtane ◽  
James G. Jackson ◽  
Yuji Ikeno ◽  
Takayoshi Ikeda ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Embryonic Development ◽  
Chromatin Remodeling ◽  
Histone Deacetylases ◽  
Globin Gene ◽  
Immunohistochemical Analysis ◽  
Wild Type ◽  
Proliferating Cells ◽  
Globin Promoter

ABSTRACT MRG15 is a highly conserved protein, and orthologs exist in organisms from yeast to humans. MRG15 associates with at least two nucleoprotein complexes that include histone acetyltransferases and/or histone deacetylases, suggesting it is involved in chromatin remodeling. To study the role of MRG15 in vivo, we generated knockout mice and determined that the phenotype is embryonic lethal, with embryos and the few stillborn pups exhibiting developmental delay. Immunohistochemical analysis indicates that apoptosis in Mrg15 − / − embryos is not increased compared with wild-type littermates. However, the number of proliferating cells is significantly reduced in various tissues of the smaller null embryos compared with control littermates. Cell proliferation defects are also observed in Mrg15 − / − mouse embryonic fibroblasts. The hearts of the Mrg15 − / − embryos exhibit some features of hypertrophic cardiomyopathy. The increase in size of the cardiomyocytes is most likely a response to decreased growth of the cells. Mrg15 − / − embryos appeared pale, and microarray analysis revealed that α-globin gene expression was decreased in null versus wild-type embryos. We determined by chromatin immunoprecipitation that MRG15 was recruited to the α-globin promoter during dimethyl sulfoxide-induced mouse erythroleukemia cell differentiation. These findings demonstrate that MRG15 has an essential role in embryonic development via chromatin remodeling and transcriptional regulation.

Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells

1990 ◽  
Vol 10 (12) ◽  
pp. 6755-6758
Author(s):  
B R Stanton ◽  
S W Reid ◽  
L F Parada
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Homologous Recombination ◽  
Embryonic Development ◽  
Cell Lines ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Germ Line Transmission

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.

Brachyury - a gene affecting mouse gastrulation and early organogenesis

Development ◽  
1992 ◽  
Vol 116 (Supplement) ◽  
pp. 157-165 ◽  
Author(s):  
R. S. P. Beddington ◽  
P. Rashbass ◽  
V. Wilson
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Coat Colour ◽  
Es Cell ◽  
Wild Type ◽  
Mesoderm Cell ◽  
Rostrocaudal Axis

Mouse embryos that are homozygous for the Brachyury (T) deletion die at mid-gestation. They have prominent defects in the notochord, the allantois and the primitive streak. Expression of the T gene commences at the onset of gastrulation and is restricted to the primitive streak, mesoderm emerging from the streak, the head process and the notochord. Genetic evidence has suggested that there may be an increasing demand for T gene function along the rostrocaudal axis. Experiments reported here indicate that this may not be the case. Instead, the gradient in severity of the T defect may be caused by defective mesoderm cell movements, which result in a progressive accumulation of mesoderm cells near the primitive streak. Embryonic stem (ES) cells which are homozygous for the T deletion have been isolated and their differentiation in vitro and in vivo compared with that of heterozygous and wild-type ES cell lines. In +/+ ↔ T/T ES cell chimeras the Brachyury phenotype is not rescued by the presence of wild-type cells and high level chimeras show most of the features characteristic of intact T/T mutants. A few offspring from blastocysts injected with T/T ES cells have been born, several of which had greatly reduced or abnormal tails. However, little or no ES cell contribution was detectable in these animals, either as coat colour pigmentation or by isozyme analysis. Inspection of potential +/+ ↔ T/T ES cell chimeras on the 11th or 12th day of gestation, stages later than that at which intact T/T mutants die, revealed the presence of chimeras with caudal defects. These chimeras displayed a gradient of ES cell colonisation along the rostrocaudal axis with increased colonisation of caudal regions. In addition, the extent of chimerism in ectodermal tissues (which do not invaginate during gastrulation) tended to be higher than that in mesodermal tissues (which are derived from cells invaginating through the primitive streak). These results suggest that nascent mesoderm cells lacking the T gene are compromised in their ability to move away from the primitive streak. This indicates that one function of the T genemay be to regulate cell adhesion or cell motility properties in mesoderm cells. Wild-type cells in +/+ ↔ T/T chimeras appear to move normally to populate trunk and head mesoderm, suggesting that the reduced motility in T/T cells is a cell autonomous defect

scholarly journals Mast Cell-Specific Expression of Human Siglec-8 in Conditional Knock-in Mice

2018 ◽  
Vol 20 (1) ◽  
pp. 19 ◽  
Author(s):  
Yadong Wei ◽  
Krishan Chhiba ◽  
Fengrui Zhang ◽  
Xujun Ye ◽  
Lihui Wang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mast Cell ◽  
Mast Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Surface Protein ◽  
Cell Types ◽  
Mouse Strains ◽  
Specific Expression

Sialic acid-binding Ig-like lectin 8 (Siglec-8) is expressed on the surface of human eosinophils, mast cells, and basophils—cells that participate in allergic and other diseases. Ligation of Siglec-8 by specific glycan ligands or antibodies triggers eosinophil death and inhibits mast cell degranulation; consequences that could be leveraged as treatment. However, Siglec-8 is not expressed in murine and most other species, thus limiting preclinical studies in vivo. Based on a ROSA26 knock-in vector, a construct was generated that contains the CAG promoter, a LoxP-floxed-Neo-STOP fragment, and full-length Siglec-8 cDNA. Through homologous recombination, this Siglec-8 construct was targeted into the mouse genome of C57BL/6 embryonic stem (ES) cells, and chimeric mice carrying the ROSA26-Siglec-8 gene were generated. After cross-breeding to mast cell-selective Cre-recombinase transgenic lines (CPA3-Cre, and Mcpt5-Cre), the expression of Siglec-8 in different cell types was determined by RT-PCR and flow cytometry. Peritoneal mast cells (dual FcεRI+ and c-Kit+) showed the strongest levels of surface Siglec-8 expression by multicolor flow cytometry compared to expression levels on tissue-derived mast cells. Siglec-8 was seen on a small percentage of peritoneal basophils, but not other leukocytes from CPA3-Siglec-8 mice. Siglec-8 mRNA and surface protein were also detected on bone marrow-derived mast cells. Transgenic expression of Siglec-8 in mice did not affect endogenous numbers of mast cells when quantified from multiple tissues. Thus, we generated two novel mouse strains, in which human Siglec-8 is selectively expressed on mast cells. These mice may enable the study of Siglec-8 biology in mast cells and its therapeutic targeting in vivo.

scholarly journals Differences in Gene Expression between Wild Type and Hoxa1 Knockout Embryonic Stem Cells after Retinoic Acid Treatment or Leukemia Inhibitory Factor (LIF) Removal

2005 ◽  
Vol 280 (16) ◽  
pp. 16484-16498 ◽  
Author(s):  
Eduardo Martinez-Ceballos ◽  
Pierre Chambon ◽  
Lorraine J. Gudas
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Retinoic Acid ◽  
Leukemia Inhibitory Factor ◽  
Target Genes ◽  
Hox Genes ◽  
Es Cells ◽  
Embryonic Stem ◽  
Inhibitory Factor ◽  
Hoxa Cluster

Homeobox (Hox) genes encode a family of transcription factors that regulate embryonic patterning and organogenesis. In embryos, alterations of the normal pattern of Hox gene expression result in homeotic transformations and malformations. Disruption of theHoxa1gene, the most 3′ member of the Hoxa cluster and a retinoic acid (RA) direct target gene, results in abnormal ossification of the skull, hindbrain, and inner ear deficiencies, and neonatal death. We have generated Hoxa1-/-embryonic stem (ES) cells (named Hoxa1-15) from Hoxa1-/-mutant blastocysts to study the Hoxa1 signaling pathway. We have characterized in detail these Hoxa1-/-ES cells by performing microarray analyses, and by this technique we have identified a number of putative Hoxa-1 target genes, including genes involved in bone development (e.g. Col1a1,Postn/Osf2, and the bone sialoprotein gene orBSP), genes that are expressed in the developing brain (e.g. Nnat,Wnt3a,BDNF,RhoB, andGbx2), and genes involved in various cellular processes (e.g. M-RAS,Sox17,Cdkn2b,LamA1,Col4a1,Foxa2,Foxq1,Klf5, andIgf2). Cell proliferation assays and Northern blot analyses of a number of ES cell markers (e.g. Rex1,Oct3/4,Fgf4, andBmp4) suggest that the Hoxa1 protein plays a role in the inhibition of cell proliferation by RA in ES cells. Additionally, Hoxa1-/-ES cells express high levels of various endodermal markers, includingGata4andDab2, and express much lessFgf5after leukemia inhibitory factor (LIF) withdrawal. Finally, we propose a model in which the Hoxa1 protein mediates repression of endodermal differentiation while promoting expression of ectodermal and mesodermal characteristics.

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