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.

scholarly journals ZC3H4—a novel Cys-Cys-Cys-His-type zinc finger protein—is essential for early embryogenesis in mice†

2020 ◽  
Author(s):  
Jianmin Su ◽  
Xiaosu Miao ◽  
Danielle Archambault ◽  
Jesse Mager ◽  
Wei Cui
Keyword(s):  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Inner Cell Mass ◽  
Functional Characterization ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Number ◽  
Dna Breaks ◽  
Finger Protein

Abstract Zinc finger domains of the Cys-Cys-Cys-His (CCCH) class are evolutionarily conserved proteins that bind nucleic acids and are involved in various biological processes. Nearly 60 CCCH-type zinc finger proteins have been identified in humans and mice, most have not been functionally characterized. Here, we provide the first in vivo functional characterization of ZC3H4—a novel CCCH-type zinc finger protein. Our results show that although Zc3h4 mutant embryos exhibit normal morphology at E3.5 blastocyst stage, they cannot be recovered at E7.5 early post-gastrulation stage, suggesting implantation failure. Outgrowth assays reveal that mutant blastocysts either fail to hatch from the zona pellucida, or can hatch but do not form a typical inner cell mass colony, the source of embryonic stem cells (ESCs). Although there is no change in levels of reactive oxygen species, Zc3h4 mutants display severe DNA breaks and reduced cell proliferation. Analysis of lineage specification reveals that both epiblast and primitive endoderm lineages are compromised with severe reductions in cell number and/or specification in the mutant blastocysts. In summary, these findings demonstrate the essential role of ZC3H4 during early mammalian embryogenesis.

102. THE EFFECT OF INSULIN ON EMBRYONIC STEM CELL PROGENITOR CELLS IN THE MOUSE BLASTOCYST

2009 ◽  
Vol 21 (9) ◽  
pp. 21
Author(s):  
J. M. Campbell ◽  
I. Vassiliev ◽  
M. B. Nottle ◽  
M. Lane
Keyword(s):  
Progenitor Cells ◽  
Cell Fate ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Number ◽  
Human Embryos ◽  
Cell Stage ◽  
Stage Of Development

Human ESCs are produced from embryos donated at the mid-stage of pre-implantation development. This cryostorage reduced viability. However, it has been shown that this can be improved by the addition of growth factors to culture medium. The aim of the present study was to examine whether the addition of insulin to embryo culture medium from the 8-cell stage of development increases the number of ES cell progenitor cells in the epiblast in a mouse model. In vivo produced mouse zygotes (C57Bl6 strain) were cultured in G1 medium for 48h to the 8-cell stage, followed by culture in G2 supplemented with insulin (0, 0.17, 1.7 and 1700pM) for 68h, at 37 o C , in 5% O2, 6%CO2, 89% N2 . The number of cells in the inner cell mass (ICM) and epiblast was determined by immunohistochemical staining for Oct4 and Nanog. ICM cells express Oct4, epiblast cells express both Oct4 and Nanog. The addition of insulin at the concentrations examined did not increase the ICM. However, at 1.7pM insulin increased the number of epiblast cells (6.6±0.5 cells vs 4.1±0.5, P=0.001) in the ICM, which increased the proportion of the ICM that was epiblast (38.9±3.7% compared to 25.8±3.4% in the control P=0.01). This indicates that the increase in the epiblast is brought about by a shift in cell fate as opposed to an increase in cell division. The effect of insulin on the proportion of cells in the epiblast was investigated using inhibitors of phosphoinositide3-kinase (PI3K) (LY294002, 50µM); one of insulin's main second messengers, and p53 (pifithrin-α, 30µg/ml); a pro-apoptotic protein inactivated by PI3K. Inhibition of PI3K eliminated the increase caused by insulin (4.5±0.3 cells versus 2.2±0.3 cells, P<0.001), while inhibition of p53 increased the epiblast cell number compared to the control (7.1±0.8 and 4.1±0.7 respectively P=0.001). This study shows that insulin increases epiblast cell number through the activation of PI3K and the inhibition of p53, and may be a strategy for improving ESC isolation from human embryos.

Establishment of rat embryonic stem-like cells from the morula using a combination of feeder layers

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 229-237 ◽  
Author(s):  
Chiaki Sano ◽  
Asako Matsumoto ◽  
Eimei Sato ◽  
Emiko Fukui ◽  
Midori Yoshizawa ◽  
...  
Keyword(s):  
Cell Lines ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Preimplantation Embryos ◽  
Long Term Culture ◽  
Oct4 Expression ◽  
Feeder Layers

SummaryEmbryonic stem (ES) cells are characterized by pluripotency, in particular the ability to form a germline on injection into blastocysts. Despite numerous attempts, ES cell lines derived from rat embryos have not yet been established. The reason for this is unclear, although certain intrinsic biological differences among species and/or strains have been reported. Herein, using Wistar-Imamichi rats, specific characteristics of preimplantation embryos are described. At the blastocyst stage, Oct4 (also called Pou5f1) was expressed in both the inner cell mass (ICM) and the trophectoderm (TE), whereas expression of Cdx2 was localized to the TE. In contrast, at an earlier stage, expression of Oct4 was detected in all the nuclei in the morula. These stages were examined using a combination of feeder layers (rat embryonic fibroblast [REF] for primary outgrowth and SIM mouse embryo-derived thioguanine- and ouabain-resistant [STO] cells for passaging) to establish rat ES-like cell lines. The rat ES-like cell lines obtained from the morula maintained expression of Oct4 over long-term culture, whereas cell lines derived from blastocysts lost pluripotency during early passage. The morula-derived ES-like cell lines showed Oct4 expression in a long-term culture, even after cryogenic preservation, thawing and EGFP transfection. These results indicate that rat ES-like cell lines with long-term Oct4 expression can be established from the morula of Wistar-Imamichi rats using a combination of feeder layers.

scholarly journals Controlling embryonic stem cell proliferation and pluripotency: the role of PI3K- and GSK-3-dependent signalling

2011 ◽  
Vol 39 (2) ◽  
pp. 674-678 ◽  
Author(s):  
Melanie J. Welham ◽  
Emmajayne Kingham ◽  
Yolanda Sanchez-Ripoll ◽  
Benjamin Kumpfmueller ◽  
Michael Storm ◽  
...  
Keyword(s):  
Cell Fate ◽  
Molecular Mechanisms ◽  
Inner Cell Mass ◽  
Glycogen Synthase ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Types ◽  
Specific Cell ◽  
Human Escs ◽  
Phosphoinositide 3 Kinase

ESCs (embryonic stem cells) are derived from the inner cell mass of pre-implantation embryos and are pluripotent, meaning they can differentiate into all of the cells that make up the adult organism. This property of pluripotency makes ESCs attractive as a model system for studying early development and for the generation of specific cell types for use in regenerative medicine and drug screening. In order to harness their potential, the molecular mechanisms regulating ESC pluripotency, proliferation and differentiation (i.e. cell fate) need to be understood so that pluripotency can be maintained during expansion, while differentiation to specific lineages can be induced accurately when required. The present review focuses on the potential roles that PI3K (phosphoinositide 3-kinase) and GSK-3 (glycogen synthase kinase 3)-dependent signalling play in the co-ordination and integration of mouse ESC pluripotency and proliferation and contrast this with our understanding of their functions in human ESCs.

scholarly journals Human ES Cell Culture Conditions Fail to Preserve the Mouse Epiblast State

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
A. S. Devika ◽  
Anna Montebaur ◽  
S. Saravanan ◽  
Raghu Bhushan ◽  
Frederic Koch ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Culture Condition ◽  
Prolonged Exposure ◽  
Inner Cell Mass ◽  
Species Difference ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Preimplantation Embryos

Mouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) are the pluripotent stem cells (PSCs), derived from the inner cell mass (ICM) of preimplantation embryos at embryonic day 3.5 (E3.5) and postimplantation embryos at E5.5-E7.5, respectively. Depending on their environment, PSCs can exist in the so-called naïve (ESCs) or primed (EpiSCs) states. Exposure to EpiSC or human ESC (hESC) culture condition can convert mESCs towards an EpiSC-like state. Here, we show that the undifferentiated epiblast state is however not stabilized in a sustained manner when exposing mESCs to hESC or EpiSC culture condition. Rather, prolonged exposure to EpiSC condition promotes a transition to a primitive streak- (PS-) like state via an unbiased epiblast-like intermediate. We show that the Brachyury-positive PS-like state is likely promoted by endogenous WNT signaling, highlighting a possible species difference between mouse epiblast-like stem cells and human Embryonic Stem Cells.

scholarly journals BRG1 GovernsNanogTranscription in Early Mouse Embryos and Embryonic Stem Cells via Antagonism of Histone H3 Lysine 9/14 Acetylation

2015 ◽  
Vol 35 (24) ◽  
pp. 4158-4169 ◽  
Author(s):  
Timothy S. Carey ◽  
Zubing Cao ◽  
Inchul Choi ◽  
Avishek Ganguly ◽  
Catherine A. Wilson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Histone H3 ◽  
Embryonic Stem ◽  
Histone Deacetylation ◽  
Preimplantation Embryos ◽  
Nucleosome Remodeling ◽  
Histone Deacetylase 1

During mouse preimplantation development, the generation of the inner cell mass (ICM) and trophoblast lineages comprises upregulation ofNanogexpression in the ICM and its silencing in the trophoblast. However, the underlying epigenetic mechanisms that differentially regulateNanogin the first cell lineages are poorly understood. Here, we report that BRG1 (Brahma-related gene 1) cooperates with histone deacetylase 1 (HDAC1) to regulateNanogexpression. BRG1 depletion in preimplantation embryos andCdx2-inducible embryonic stem cells (ESCs) revealed that BRG1 is necessary forNanogsilencing in the trophoblast lineage. Conversely, in undifferentiated ESCs, loss of BRG1 augmentedNanogexpression. Analysis of histone H3 within theNanogproximal enhancer revealed that H3 lysine 9/14 (H3K9/14) acetylation increased in BRG1-depleted embryos and ESCs. Biochemical studies demonstrated that HDAC1 was present in BRG1-BAF155 complexes and BRG1-HDAC1 interactions were enriched in the trophoblast lineage. HDAC1 inhibition triggered an increase in H3K9/14 acetylation and a corresponding rise inNanogmRNA and protein, phenocopying BRG1 knockdown embryos and ESCs. Lastly, nucleosome-mapping experiments revealed that BRG1 is indispensable for nucleosome remodeling at theNanogenhancer during trophoblast development. In summary, our data suggest that BRG1 governsNanogexpression via a dual mechanism involving histone deacetylation and nucleosome remodeling.

scholarly journals Depletion of Demethylase KDM6 Enhances Early Neuroectoderm Commitment of Human PSCs

2021 ◽  
Vol 9 ◽  
Author(s):  
Yajing Meng ◽  
Tianzhe Zhang ◽  
Ran Zheng ◽  
Song Ding ◽  
Jie Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Fate ◽  
Cell Lines ◽  
Histone Methylation ◽  
Pluripotent Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Neural Induction

Epigenetic modifications play a crucial role in neurogenesis, learning, and memory, but the study of their role in early neuroectoderm commitment from pluripotent inner cell mass is relatively lacking. Here we utilized the system of directed neuroectoderm differentiation from human embryonic stem cells and identified that KDM6B, an enzyme responsible to erase H3K27me3, was the most upregulated enzyme of histone methylation during neuroectoderm differentiation by transcriptome analysis. We then constructed KDM6B-null embryonic stem cells and found strikingly that the pluripotent stem cells with KDM6B knockout exhibited much higher neuroectoderm induction efficiency. Furthermore, we constructed a series of embryonic stem cell lines knocking out the other H3K27 demethylase KDM6A, and depleting both KDM6A and KDM6B, respectively. These cell lines together confirmed that KDM6 impeded early neuroectoderm commitment. By RNA-seq, we found that the expression levels of a panel of WNT genes were significantly affected upon depletion of KDM6. Importantly, the result that WNT agonist and antagonist could abolish the differential neuroectoderm induction due to manipulating KDM6 further demonstrated that WNT was the major downstream of KDM6 during early neural induction. Moreover, we found that the chemical GSK-J1, an inhibitor of KDM6, could enhance neuroectoderm induction from both embryonic stem cells and induced pluripotent stem cells. Taken together, our findings not only illustrated the important role of the histone methylation modifier KDM6 in early neurogenesis, providing insights into the precise epigenetic regulation in cell fate determination, but also showed that the inhibitor of KDM6 could facilitate neuroectoderm differentiation from human pluripotent stem cells.

scholarly journals Alpha-ketoglutarate affects murine embryo development through metabolic and epigenetic modulations

Reproduction ◽  
2019 ◽  
Vol 158 (2) ◽  
pp. 125-135 ◽  
Author(s):  
Zhenzhen Zhang ◽  
Changjiu He ◽  
Lu Zhang ◽  
Tianqi Zhu ◽  
Dongying Lv ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Embryo Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Dna Demethylation ◽  
Basic Knowledge ◽  
Preimplantation Embryos ◽  
Foetal Growth

α-Ketoglutarate (α-KG) is an intermediary metabolite in the tricarboxylic acid (TCA) cycle and functions to inhibit ATPase and maintain the pluripotency of embryonic stem cells (ESCs); however, little is known regarding the effects of α-KG on the development of preimplantation embryos. Herein, we report that α-KG (150 μM) treatment significantly promoted the blastocyst rate, the number of inner cell mass (ICM) cells and foetal growth after embryo transfer. Mechanistic studies revealed two important pathways involved in the α-KG effects on embryo development. First, α-KG modulates mitochondria function by inducing relatively low ATP production without modification of mitochondrial copy number. The relatively low energy metabolism preserves the pluripotency and competence of the ICM. Second, α-KG modifies epigenetics in embryos cultured in vitro by affecting the activity of the DNA demethylation enzyme TET and the DNA methylation gene Dnmt3a to increase the ratio of 5hmC/5mC ratio. Elevation of the 5hmC/5mC ratio not only promotes the pluripotency of the ICM but also leads to a methylation level in an in vitro embryo close to that in an in vivo embryo. All these functions of α-KG collectively contribute to an increase in the number of ICM cells, leading to greater adaptation of cultured embryos to in vitro conditions and promoting foetal growth after embryo transfer. Our findings provide basic knowledge regarding the mechanisms by which α-KG affects embryo development and cell differentiation.

89 INHIBITION OF MAPKK AND GSK3 SIGNALLING PROMOTES DEVELOPMENT AND EPIBLAST-SPECIFIC EXPRESSION OF PLURIPOTENCY MARKERS IN BOVINE BLASTOCYSTS

2013 ◽  
Vol 25 (1) ◽  
pp. 192
Author(s):  
D. Harris ◽  
B. Huang ◽  
B. Oback
Keyword(s):  
Dimethyl Sulfoxide ◽  
Inner Cell Mass ◽  
Glycogen Synthase ◽  
Cell Mass ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Mitogen Activated Protein Kinase ◽  
Differential Staining ◽  
Blastocyst Development ◽  
Specific Expression

During blastocyst development, the inner cell mass segregates into the epiblast and the hypoblast. These 2 tissues form morphologically and molecularly distinct cell populations that subsequently develop into the embryo proper and some extraembryonic components, respectively. In mouse, isolated epiblast cells can be directly converted into pluripotent embryonic stem cells, capable of differentiating into all cell types of an adult animal. Epiblast pluripotency is promoted by pharmacological inhibition of mitogen-activated protein kinase kinase (Mapkk). This shields epiblast cells from secreted fibroblast growth factor (Fgf), which would otherwise instruct them to exit pluripotency and differentiate into extraembryonic lineages. Indirect stimulation of the Wnt pathway by inhibiting glycogen synthase kinase 3 (GSK3) further antagonises inductive Fgf/Mapkk signalling. Thus the double inhibition (2i) of Mapkk and Gsk3 effectively promotes pluripotency (Q. L. Ying et al. 2008 Nature 453, 519–523; J. Nichols et al. 2009 Development 136, 3215–3222). We investigated the effect of 2i culture on bovine blastocysts. The IVF embryos were cultured in the presence of dimethyl sulfoxide or inhibitors of MAPKK (0.4 µM PD0325901) and GSK3 (3 µM CHIR99021) from the zygote (Day 1) stage onward. Compared to vehicle controls, 2i increased the abundance of cumulus cells in bovine IVF cultures, compromising blastocyst formation in cumulus-intact (248/823 = 30% v. 211/824 = 26%, respectively, n = 10; P < 0.05) but not cumulus-free cultures (546/1653 = 33% v. 572/1674 = 34%, respectively, n = 15; P = 0.51). In all subsequent experiments, we therefore cultured cumulus-free zygotes in 2i v. dimethyl sulfoxide until the blastocyst stage. This treatment increased the proportion of hatching (19/433 = 4% v. 7/416 = 2%, respectively, n = 10; P < 0.05) at the expense of early blastocysts (70/433 = 16% v. 93/416 = 22%, respectively, n = 11; P < 0.05). Differential staining of expanded IETS grade 1 and 2 blastocysts showed that 2i culture increased putative inner cell mass, trophectoderm, and total cell nuclei numbers by about 30% compared with controls (57 v. 43, 89 v. 69, and 146 v. 112, respectively; P < 0.01). Accelerated development and increased cell numbers were accompanied by gene expression changes in grade 1 and 2 blastocysts. Under 2i conditions, mRNA abundance of putative epiblast markers NANOG and SOX2 was >3-fold increased (P < 0.0001 and P < 0.01, respectively), and the putative hypoblast marker GATA4 was 2-fold reduced (P < 0.05). Other lineage-related markers (POU5F1, KLF4, DPPA3, and CDX2) showed no significant changes. Using microsurgical blastocyst dissection, we found that the increase in NANOG and SOX2 levels was specific to the inner cell mass-containing portion (7-fold for NANOG and 3-fold for SOX2; P < 0.00005 and P < 0.05, respectively) and not due to ectopic expression in the trophoblast-containing part, which showed similarly low expression levels for both genes. In summary, 2i treatment primed bovine blastocysts for pluripotency in the epiblast. Supported by MSI C10X1002.

scholarly journals YY2 in Mouse Preimplantation Embryos and in Embryonic Stem Cells

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1123
Author(s):  
Raquel Pérez-Palacios ◽  
María Climent ◽  
Javier Santiago-Arcos ◽  
Sofía Macías-Redondo ◽  
Martin Klar ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Yin Yang

Yin Yang 2 encodes a mammalian-specific transcription factor (YY2) that shares high homology in the zinc finger region with both YY1 and REX1/ZFP42, encoded by the Yin Yang 1 and Reduced Expression Protein 1/Zinc Finger Protein 42 gene, respectively. In contrast to the well-established roles of the latter two in gene regulation, X chromosome inactivation and binding to specific transposable elements (TEs), much less is known about YY2, and its presence during mouse preimplantation development has not been described. As it has been reported that mouse embryonic stem cells (mESC) cannot be propagated in the absence of Yy2, the mechanistic understanding of how Yy2 contributes to mESC maintenance remains only very partially characterized. We describe Yy2 expression studies using RT-PCR and staining with a high-affinity polyclonal serum in mouse embryos and mESC. Although YY2 is expressed during preimplantation development, its presence appears dispensable for developmental progress in vitro until formation of the blastocyst. Attenuation of Yy2 levels failed to alter either Zscan4 levels in two-cell embryos or IAP and MERVL levels at later preimplantation stages. In contrast to previous claims that constitutively expressed shRNA against Yy2 in mESC prohibited the propagation of mESC in culture, we obtained colonies generated from mESC with attenuated Yy2 levels. Concomitant with a decreased number of undifferentiated colonies, Yy2-depleted mESC expressed higher levels of Zscan4 but no differences in the expression of TEs or other pluripotency markers including Sox2, Oct4, Nanog and Esrrb were observed. These results confirm the contribution of Yy2 to the maintenance of mouse embryonic stem cells and show the preimplantation expression of YY2. These functions are discussed in relation to mammalian-specific functions of YY1 and REX1.

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