scholarly journals 105 EXPRESSION OF PLURIPOTENCY-DETERMINING FACTORS Oct-4 AND NANOG IN PRE-IMPLANTATION GOAT EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 203 ◽  
Author(s):  
S. He ◽  
D. Pant ◽  
S. Bischoff ◽  
W. Gavin ◽  
D. Melican ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Mrna Expression ◽  
Cdna Sequence ◽  
Inner Cell Mass ◽  
Protein Localization ◽  
Expression Patterns ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Pcr Product ◽  
Determining Factors

The objective of this study was to determine the expression patterns of the pluripotency-determining factors, Oct-4 and Nanog, in pre-implantation goat embryos. The POU octamer-binding domain transcription factor Oct-4 and the homeobox transcription factor Nanog have been shown to play key roles in the maintenance of pluripotency in the inner cell mass (ICM) of pre-implantation mouse embryos and in embryonic stem cells. As Oct-4 protein has been observed in human, monkey, bovine, and porcine pre-implantation embryos, its role in embryonic development and differentiation may be conserved across these species. The patterns of mRNA expression for Oct-4 and Nanog have not been reported for ruminant embryos. In this study, total RNA was extracted from 10 in vivo-derived goat embryos at each stage (8-cell, morula, and blastocyst) using an Absolutely RNA Nanoprep Kit (Stratagene, La Jolla, CA, USA). The first-strand cDNAs were synthesized using Superscript III (Invitrogen, Carlsbad, CA, USA) and cDNAs were amplified with PfuUltra hotstart PCR master mix (Stratagene). Oct-4 primers were designed based on bovine Oct-4 open-reading sequence, while Nanog primers were designed based on the human Nanog open-reading sequence. Expression screening by PCR was performed. Oct-4 mRNA expression was detected at the 8-cell, morula and blastocyst stages. Sequencing of the 1.1-kb PCR product with Oct-4 primers revealed 87% homology to human cDNA sequence and 96% homology to the bovine sequence. Protein localization of Oct-4 as observed by immunocytochemistry was diffuse at the morula stage, but moved to a more nuclear location at the blastocyst stage. Oct-4 protein and mRNA expression were detected in both the ICM and trophectoderm of expanded blastocysts. This pattern of protein expression is similar to that reported by others in the pig and cow. As caprine, bovine, and porcine embryos all show extensive proliferation and elongation of the trophectoderm, continued expression of Oct-4 protein in the trophectoderm may be necessary to prevent premature differentiation of the trophectoderm. Nanog mRNA was detected at the morula and blastocyst stages. Nanog mRNA was detected in the ICM but not the trophectoderm of expanded goat blastocysts, a pattern that follows the expression observed in mice. Sequencing of the 698 bp PCR product obtained by RT-PCR from goat blastocysts confirmed that the mRNA detected was Nanog. Sequence alignment (ClustalW) showed that the cDNA sequence identities were 96% between goat and human and 70% between goat and mouse. The amino acid identities were 93% between goat and human and 52% between goat and mouse. To our knowledge this is the first report of detection of Nanog in domestic animals. These results are supportive of the premise that core components involved in the control of pluripotency are analogous across vertebrate species.

scholarly journals 246 EXPRESSION PATTERN OF NANOG AND Par3 GENES IN IN VITRO-DERIVED BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 231 ◽  
Author(s):  
F. Gandolfi ◽  
F. Cillo ◽  
S. Antonini ◽  
S. Colleoni ◽  
I. Lagutina ◽  
...  
Keyword(s):  
Cell Fate ◽  
Cdna Sequence ◽  
Inner Cell Mass ◽  
Expression Patterns ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Genome Activation

Homeobox genes have been demonstrated to be important in patterning and lineage specification during early embryogenesis. Nanog belongs to the family of DNA-binding transcription factors and has been shown to maintain pluripotency of embryonic stem cells, both in murine and human. Par3 plays an essential role in determining cell fate of the early mouse embryo, leading to the generation of the inner cell mass and the trophectoderm. No information is available on these genes in the bovine; therefore, the aim of the present study was to identify and characterize Nanog and Par3 expression in bovine embryos. Oocytes recovered from slaughterhouse ovaries were matured for 22 h, fertilized in vitro and then cultured in mSOFaa medium. RNA was extracted from pools of five oocytes and embryos at different stages of development (2-, 4-, 8-, 16-cell, morula and blastocyst). It was then reverse transcribed, and PCR runs were carried out with primers specifically designed for Nanog and Par3, based on the sequence data bank available. The amplified products were separated on a 2% TAE agarose gel, purified, sequenced and aligned using Clustal W. Comparison of the bovine Nanog cDNA sequence (EMBL AM039957) with databases revealed a 84% degree of homology with the human, 97% with the mouse, and 82% with the goat genes. IVF bovine embryos express Nanog only upon genome activation, becoming detectable from the 8-cell stage onward indicating that Nanog is zygotically expressed in the bovine similar to what happens in mouse, pig and goat. Bovine Par3 cDNA sequence (EMBL AM039956) shows a high degree of homology with human (83%), mouse (81%), and rat (79%). Also Par3 is expressed only upon the maternal to embryonic transition (MET) at the 8-cell stage. As opposed to the expression patterns of other early embryo genes, like Oct-4 and Zar-1, Nanog and Par3 expression patterns in bovine embryos closely resemble those described in the mouse. Since both are absent in the ooplasm and before MET, they represent useful markers for genome activation. This work was supported by FIRB RBNE01HPMX, FIRST 2004 and ESF-EuroStells.

Fgf-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse

Development ◽  
1992 ◽  
Vol 114 (3) ◽  
pp. 755-768 ◽  
Author(s):  
L. Niswander ◽  
G.R. Martin
Keyword(s):  
Tooth Development ◽  
Inner Cell Mass ◽  
Expression Patterns ◽  
Blot Hybridization ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Hybridization Analysis ◽  
Limb Bud ◽  
Northern Blot Hybridization ◽  
Tail Bud

Fgf-4, initially isolated as a transforming gene from human tumors, is a member of the Fibroblast Growth Factor (FGF) family. It has previously been shown by northern blot hybridization analysis to be expressed in teratocarcinoma and embryonic stem cells, suggesting that it plays a role in embryonic development. We have carried out an RNA in situ hybridization analysis of Fgf-4 expression in the developing mouse embryo, from fertilization through the 14th day of gestation (E14.5). Our results show that Fgf-4 RNA is first detected at the late blastocyst stage in cells that give rise to all of the embryonic lineages (inner cell mass cells). During the early stages of gastrulation, expression becomes restricted to the primitive streak where mesoderm and definitive endoderm are formed. Expression continues in the distal (rostral) two-thirds of the streak through approx. E10, and then is detected in the tail bud, which replaces the streak as the primary source of mesoderm. Additional sites of expression are found after the three primary germ layers are established and organogenesis begins. Fgf-4 RNA is detected transiently in the branchial arch units, the somitic myotome, the apical ectodermal ridge of the developing limb bud and the tooth bud, suggesting that the gene has multiple roles during embryogenesis. These results are compared with the expression patterns of other FGF genes. Taken together, the data suggest that individual members of the gene family are expressed sequentially in developmental pathways such as mesoderm formation and myogenesis, and play a role in specific epithelial-mesenchymal interactions.

scholarly journals Nuclear m6A reader YTHDC1 regulates the scaffold function of LINE1 RNA in mouse ESCs and early embryos

2021 ◽  
Author(s):  
Chuan Chen ◽  
Wenqiang Liu ◽  
Jiayin Guo ◽  
Yuanyuan Liu ◽  
Xuelian Liu ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Rrna Synthesis ◽  
Binding Ability ◽  
Transcriptome Regulation ◽  
Early Embryos ◽  
Intrinsic Mechanism ◽  
Regulatory Rnas

AbstractN6-methyladenosine (m6A) on chromosome-associated regulatory RNAs (carRNAs), including repeat RNAs, plays important roles in tuning the chromatin state and transcription, but the intrinsic mechanism remains unclear. Here, we report that YTHDC1 plays indispensable roles in the self-renewal and differentiation potency of mouse embryonic stem cells (ESCs), which highly depends on the m6A-binding ability. Ythdc1 is required for sufficient rRNA synthesis and repression of the 2-cell (2C) transcriptional program in ESCs, which recapitulates the transcriptome regulation by the LINE1 scaffold. Detailed analyses revealed that YTHDC1 recognizes m6A on LINE1 RNAs in the nucleus and regulates the formation of the LINE1-NCL partnership and the chromatin recruitment of KAP1. Moreover, the establishment of H3K9me3 on 2C-related retrotransposons is interrupted in Ythdc1-depleted ESCs and inner cell mass (ICM) cells, which consequently increases the transcriptional activities. Our study reveals a role of m6A in regulating the RNA scaffold, providing a new model for the RNA-chromatin cross-talk.

scholarly journals Differentiation of Human Embryonic Stem Cells into Neuron, Cholinergic, and Glial Cells

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Kimia Hosseini ◽  
Emilia Lekholm ◽  
Aikeremu Ahemaiti ◽  
Robert Fredriksson
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Glial Cells ◽  
Human Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cholinergic Neurons ◽  
Neuronal Cultures ◽  
Short Period

Human embryonic stem cells (hESCs) are pluripotent cells, capable of differentiation into different cellular lineages given the opportunity. Derived from the inner cell mass of blastocysts in early embryonic development, the cell self-renewal ability makes them a great tool for regenerative medicine, and there are different protocols available for maintaining hESCs in their undifferentiated state. In addition, protocols for differentiation into functional human neural stem cells (hNSCs), which have the potential for further differentiation into various neural cell types, are available. However, many protocols are time-consuming and complex and do not always fit for purpose. In this study, we carefully combined, optimized, and developed protocols for differentiation of hESCs into adherent monolayer hNSCs over a short period of time, with the possibility of both expansion and freezing. Moreover, the method details further differentiation into neurons, cholinergic neurons, and glial cells in a simple, single step by step protocol. We performed immunocytochemistry, qPCR, and electrophysiology to examine the expression profile and characteristics of the cells to verify cell lineage. Using presented protocols, the creation of neuronal cultures, cholinergic neurons, and a mixed culture of astrocytes and oligodendrocytes can be completed within a three-week time period.

The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes

1993 ◽  
Vol 13 (12) ◽  
pp. 7971-7976
Author(s):  
L M Whyatt ◽  
A Düwel ◽  
A G Smith ◽  
P D Rathjen
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Expression System ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Expression Vectors ◽  
Developmental Control ◽  
Developmental Potential

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.

scholarly journals Transcription Factor Lbx1 Expression in Mouse Embryonic Stem Cell-Derived Phenotypes

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Stefanie Schmitteckert ◽  
Cornelia Ziegler ◽  
Liane Kartes ◽  
Alexandra Rolletschek
Keyword(s):  
Transcription Factor ◽  
Developmental Stages ◽  
Troponin T ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Cardiac Cells ◽  
Cell Stage

Transcription factor Lbx1 is known to play a role in the migration of muscle progenitor cells in limb buds and also in neuronal determination processes. In addition, involvement of Lbx1 in cardiac neural crest-related cardiogenesis was postulated. Here, we used mouse embryonic stem (ES) cells which have the capacity to develop into cells of all three primary germ layers. Duringin vitrodifferentiation, ES cells recapitulate cellular developmental processes and gene expression patterns of early embryogenesis. Transcript analysis revealed a significant upregulation ofLbx1at the progenitor cell stage. Immunofluorescence staining confirmed the expression of Lbx1 in skeletal muscle cell progenitors and GABAergic neurons. To verify the presence of Lbx1 in cardiac cells, triple immunocytochemistry of ES cell-derived cardiomyocytes and a quantification assay were performed at different developmental stages. Colabeling of Lbx1 and cardiac specific markers troponin T, α-actinin, GATA4, and Nkx2.5 suggested a potential role in early myocardial development.

Specific expression of a retinoic acid-regulated, zinc-finger gene, Rex-1, in preimplantation embryos, trophoblast and spermatocytes

Development ◽  
1991 ◽  
Vol 113 (3) ◽  
pp. 815-824 ◽  
Author(s):  
M.B. Rogers ◽  
B.A. Hosler ◽  
L.J. Gudas
Keyword(s):  
Retinoic Acid ◽  
Germ Cell ◽  
Cell Fate ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Preimplantation Embryos ◽  
Specific Expression

We have previously isolated a cDNA clone for a gene whose expression is reduced by retinoic acid (RA) treatment of F9 embryonal carcinoma cells. The nucleotide sequence indicated that this gene, Rex-1, encodes a zinc-finger protein and thus may be a transcriptional regulator. The Rex-1 message level is high in two lines of embryonic stem cells (CCE and D3) and is reduced when D3 cells are induced to differentiate using four different growth conditions. As expected for a stem-cell-specific message, Rex-1 mRNA is present in the inner cell mass (ICM) of the day 4.5 mouse blastocyst. It is also present in the polar trophoblast of the blastocyst. One and two days later, Rex-1 message is found in the ectoplacental cone and extraembryonic ectoderm of the egg cylinder (trophoblast-derived tissues), but its abundance is much reduced in the embryonic ectoderm which is directly descended from the ICM. Rex-1 is expressed in the day 18 placenta (murine gestation is 18 days), a tissue which is largely derived from trophoblast. The only tested adult tissue that contains detectable amounts of Rex-1 mRNA is the testis. In situ hybridization and northern analyses of RNA from germ-cell-deficient mouse testis and stage-specific germ cell preparations suggest that Rex-1 expression is limited to spermatocytes (germ cells undergoing meiosis). These results suggest that Rex-1 is involved in trophoblast development and spermatogenesis, and is a useful marker for studies of early cell fate determination in the ICM.

2. Embryonic stem cells

2021 ◽  
pp. 21-37
Author(s):  
Jonathan Slack
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Practical Importance ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Human Embryos ◽  
Mouse Es Cells ◽  
Stage Embryo

‘Embryonic stem cells’ focuses on embryonic stem (ES) cells, which are grown in tissue culture from the inner cell mass of a mammalian blastocyst-stage embryo. Human ES cells offer a potential route to making the kinds of cells needed for cell therapy. ES cells were originally prepared from mouse embryos. Although somewhat different, cells grown from inner cell masses of human embryos share many properties with mouse ES cells, such as being able to grow without limit and to generate differentiated cell types. Mouse ES cells have so far been of greater practical importance than those of humans because they have enabled a substantial research industry based on the creation of genetically modified mice.

Leptin treatment of in vitro cultured embryos increases outgrowth rate of inner cell mass during embryonic stem cell derivation

2019 ◽  
Vol 55 (7) ◽  
pp. 473-481 ◽  
Author(s):  
Ali Cihan Taskin ◽  
Ahmet Kocabay ◽  
Ayyub Ebrahimi ◽  
Sercin Karahuseyinoglu ◽  
Gizem Nur Sahin ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Stem Cell Derivation

Derivation of human embryonic stem cell lines, towards clinical quality

2006 ◽  
Vol 18 (8) ◽  
pp. 823 ◽  
Author(s):  
Outi Hovatta
Keyword(s):  
Cell Lines ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Embryonic Stem ◽  
Quality Criteria ◽  
Good Manufacturing Practice ◽  
Clinical Quality ◽  
Serum Replacement ◽  
Hes Cells

Human embryonic stem (hES) cells offer an excellent source of cells for transplantation in the treatment of severe diseases. To be clinically safe, the lines have to be derived using strict quality criteria and good manufacturing practice. Animal proteins are immunogenic and may contain microbes, and they should not be used in establishing or propagating hES cells. Derivation systems have been improved towards clinical quality by establishing all 25 hES cell lines using human skin fibroblasts as feeder cells instead of mouse fibroblasts. A further 21 cell lines have been established using synthetic serum instead of fetal calf serum in the medium. In the five latest derivations, the inner cell mass was isolated mechanically instead of by immunosurgery (animal antibodies). Feeder-free derivation would be optimal, but it is not yet considered safe. Clinical-quality lines can be derived by establishing the skin fibroblast feeders in the good manufacturing practice laboratory with human serum in the medium, and by establishing the hES cells on such feeders. In this process, a serum replacement that contains only human protein can be used, the inner cell mass has to be isolated mechanically, and the colonies have to be split mechanically for passaging. Somatic cell nuclear transfer would help to overcome rejection of transplanted cells.

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