scholarly journals The Ras/Raf signaling pathway is required for progression of mouse embryos through the two-cell stage.

1994 ◽  
Vol 14 (10) ◽  
pp. 6655-6662 ◽  
Author(s):  
N Yamauchi ◽  
A A Kiessling ◽  
G M Cooper
Keyword(s):  
Monoclonal Antibody ◽  
Signaling Pathway ◽  
Antisense Oligonucleotides ◽  
Subsequent Development ◽  
Inhibitory Effect ◽  
Mouse Embryos ◽  
Dominant Negative ◽  
Cell Stage ◽  
Cell Embryo ◽  
And Function

We have used microinjection of antisense oligonucleotides, monoclonal antibody, and the dominant negative Ras N-17 mutant to interfere with Ras expression and function in mouse oocytes and early embryos. Microinjection of either ras antisense oligonucleotides or anti-Ras monoclonal antibody Y13-259 did not affect normal progression of oocytes through meiosis and arrest at metaphase II. However, microinjection of fertilized eggs with constructs expressing Ras N-17 inhibited subsequent development through the two-cell stage. The inhibitory effect of Ras N-17 was overcome by simultaneous injection of a plasmid expressing an active raf oncogene, indicating that it resulted from interference with the Ras/Raf signaling pathway. In contrast to the inhibition of two-cell embryo development resulting from microinjection of pronuclear stage eggs, microinjection of late two-cell embryos with Ras N-17 expression constructs did not affect subsequent cleavages and development to morulae and blastocysts. It thus appears that the Ras/Raf signaling pathway, presumably activated by autocrine growth factor stimulation, is specifically required at the two-cell stage, which is the time of transition between maternal and embryonic gene expression in mouse embryos.

The Ras/Raf signaling pathway is required for progression of mouse embryos through the two-cell stage

1994 ◽  
Vol 14 (10) ◽  
pp. 6655-6662
Author(s):  
N Yamauchi ◽  
A A Kiessling ◽  
G M Cooper
Keyword(s):  
Monoclonal Antibody ◽  
Signaling Pathway ◽  
Antisense Oligonucleotides ◽  
Subsequent Development ◽  
Inhibitory Effect ◽  
Mouse Embryos ◽  
Dominant Negative ◽  
Cell Stage ◽  
Cell Embryo ◽  
And Function

We have used microinjection of antisense oligonucleotides, monoclonal antibody, and the dominant negative Ras N-17 mutant to interfere with Ras expression and function in mouse oocytes and early embryos. Microinjection of either ras antisense oligonucleotides or anti-Ras monoclonal antibody Y13-259 did not affect normal progression of oocytes through meiosis and arrest at metaphase II. However, microinjection of fertilized eggs with constructs expressing Ras N-17 inhibited subsequent development through the two-cell stage. The inhibitory effect of Ras N-17 was overcome by simultaneous injection of a plasmid expressing an active raf oncogene, indicating that it resulted from interference with the Ras/Raf signaling pathway. In contrast to the inhibition of two-cell embryo development resulting from microinjection of pronuclear stage eggs, microinjection of late two-cell embryos with Ras N-17 expression constructs did not affect subsequent cleavages and development to morulae and blastocysts. It thus appears that the Ras/Raf signaling pathway, presumably activated by autocrine growth factor stimulation, is specifically required at the two-cell stage, which is the time of transition between maternal and embryonic gene expression in mouse embryos.

Distribution of microvilli on dissociated blastomeres from mouse embryos: evidence for surface polarization at compaction

Development ◽  
1981 ◽  
Vol 62 (1) ◽  
pp. 339-350
Author(s):  
W. J. D. Reeve ◽  
C. A. Ziomek
Keyword(s):  
Ligand Binding ◽  
Single Cells ◽  
Progressive Increase ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Characteristic Distribution ◽  
Surface Polarization ◽  
Cell Embryo ◽  
Fluorescent Ligand ◽  
Scanning Electron

Cells of mouse embryos develop a polarization of microvillous distribution at compaction. Cells of the 4-cell embryo show a uniform pattern of fluorescent-ligand binding and an even distribution of microvilli. Each cell of the early 8-cell embryo has a uniform distribution both of microvilli and of fluorescent ligand. During the 8-cell stage, there is a progressive increase in the incidence of cells which show microvilli restricted to a region normally on the exposed surface of the embryo. When late 8-cell embryos were disaggregated to single cells, and these sorted by pattern of fluorescent-ligand binding, each of the four patterns of staining related consistently to a characteristic distribution of microvilli as viewed by scanning electron microscopy. The 16-cell embryo possessed an inside population of uniformly labelled cells with a sparse microvillous distribution, and an outside population of cells, each of which had a microvillous pole.

Cosegregation of monoclonal antibody reactivity and cell behaviour in the mouse preimplantation embryo

Development ◽  
1982 ◽  
Vol 70 (1) ◽  
pp. 261-278
Author(s):  
Beverley J. Randle
Keyword(s):  
Monoclonal Antibody ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Antigen Expression ◽  
Cell Stage ◽  
Cell Behaviour ◽  
Minor Population ◽  
Cell Embryo ◽  
Surface Labelling

Expression of an antigen, recognized by a monoclonal antibody raised against PCI 3 embryonal carcinoma, is described in mouse preimplantation embryogenesis. The antigen is found in the cytoplasm of ovulated ova and is first noted on the cell surface of the 1-cell embryo 20 h post-ovulation. Surface labelling of blastomeres is uniform until the 8-cell stage when antigen expression becomes polarized along the radial axis of the embryo. Two major populations of blastomeres are distinguishable on division to the 16-cell morula. Dissociation of morulae in calcium-free medium yields large, polar, antigen-positive cells and small apolar cells with reduced levels of detectable antigen. A third, minor population of small, antigen-negative cells is also found in vivo. Large and small blastomeres differ in their ability to relocate within the embryo when aggregated with intact 16-cell-stage embryos. The small blastomeres of the 16-cell morula contribute significantly to the inner cell mass while the large antigen-positive cells are found only in the trophectoderm.

Quantitative aspects of RNA synthesis and polyadenylation in 1-cell and 2-cell mouse embryos

Development ◽  
1983 ◽  
Vol 74 (1) ◽  
pp. 169-182
Author(s):  
Kerry B. Clegg ◽  
Lajos Pikó
Keyword(s):  
Time Course ◽  
Specific Activity ◽  
Average Length ◽  
State Level ◽  
Mouse Embryos ◽  
High Rate ◽  
Rna Sequences ◽  
Cell Stage ◽  
New Synthesis ◽  
Cell Embryo

Mouse embryos at the late 1-cell and late 2-cell stages were labelled with [3H]adenosine for periods of up to 320 min during which the specific activity of the ATP pool was constant. The time course of the molar accumulation of adenosine was calculated for tRNA, high-molecular-weight poly(A)− RNA and poly(A) tails versus internal regions of poly(A)+ RNA. Most of the adenosine incorporation into tRNA is due to turnover of the 3′-terminal AMP but some new synthesis of tRNA also appears to take place in both 1-cell and 2-cell embryos at a rate of about 0·2 pg/embryo/h. In the poly(A)- RNA fraction, an unstable component which is assumed to be heterogeneous nuclear RNA is synthesized at a high rate and accumulates at a steady-state level of about 1·5 pg/embryo in the 1-cell embryo and about 3·0 pg/embryo in the 2-cell embryo. Both 1-cell and 2-cell embryos synthesize relatively stable heterogeneous poly(A)− RNA, assumed to be mRNA, at a rate of about 0·3 pg/embryo/h; 2-cell embryos also synthesize mature ribosomal RNA at a rate of about 0·4 pg/embryo·h. Internally labelled poly(A)+ RNA is synthesized at a low rate in the 1-cell embryo, about 0·045 pg/embryo/h, but the rate increases to about 0·2 pg/embryo/h by the 2-cell stage. A striking feature of the 1-cell embryo is the high rate of synthesis of poly(A) tails, about 2·5 × 106 tails/embryo/h of an average length of (A)43, due almost entirely to cytoplasmic polyadenylation. This and other evidence suggests a turnover of the poly(A)+ RNA population in 1-cell embryos as a result of polyadenylation of new RNA sequences and degradation of some of the pre-existing poly(A)+ RNA. In the 2-cell embryo, the rate of synthesis of poly(A) tails (average length (A)93) is estimated at about 0·8 × 106tails/embryo/h and a significant fraction of poly(A) synthesis appears to be nuclear.

scholarly journals Efficient generation of targeted large insertions in mouse embryos using 2C-HR-CRISPR

2017 ◽  
Author(s):  
Bin Gu ◽  
Eszter Posfai ◽  
Janet Rossant
Keyword(s):  
Cell Fate ◽  
Protein Function ◽  
Rapid Assessment ◽  
Mouse Genetics ◽  
Mouse Embryos ◽  
Open Chromatin ◽  
Cell Stage ◽  
Cell Fate Decisions ◽  
Efficient Generation ◽  
Cell Embryo

Rapid and efficient generation of large fragment targeted knock-in mouse models is still a major hurdle in mouse genetics. Here we developed 2C-HR-CRISPR, a highly efficient gene editing method based on introducing CRISPR reagents into mouse embryos at the 2-cell stage, taking advantage of the likely increase in HR efficiency during the long G2 phase and open chromatin structure of the 2-cell embryo. With 2C-HR-CRISPR and a modified biotin-streptavidin approach to localize repair templates to target sites, we rapidly targeted 20 endogenous genes that are expressed in mouse blastocysts with fluorescent reporters and generated reporter mouse lines. We showcase the first live triple-color blastocyst with all three lineages differentially reported. Additionally, we demonstrated efficient double targeting, enabling rapid assessment of the auxin-inducible degradation system for probing protein function in mouse embryos. These methods open up exciting avenues for exploring cell fate decisions in the blastocyst and later stages of development. We also suggest that 2C-HR-CRISPR can be a better alternative to random transgenesis by ensuring transgene insertions at defined ‘safe harbor’ sites.

The effects of embryo stage and cell number on the composition of mouse aggregation chimaeras

Zygote ◽  
2000 ◽  
Vol 8 (3) ◽  
pp. 235-243 ◽  
Author(s):  
Pin-chi Tang ◽  
John D. West
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Advanced Embryo ◽  
Preimplantation Mouse Embryos ◽  
Preimplantation Mouse ◽  
Cell Embryo ◽  
Embryo Stage

Studies with intact preimplantation mouse embryos and some types of chimaeric aggregates have shown that the most advanced cells are preferentially allocated to the inner cell mass (ICM) rather than the trophectoderm. Thus, differences between 4-cell and 8-cell stage embryos could contribute to the tendency for tetraploid cells to colonise the trophectoderm more readily than the ICM in 4-cell tetraploid[harr ]8 cell diploid chimaeras. The aim of the present study was to test whether 4-cell stage embryos in 4-cell diploid[harr ]8-cell diploid aggregates contributed equally to all lineages present in the E12.5 conceptus. These chimaeras were compared with those produced from standard aggregates of two whole 8-cell embryos and aggregates of half an 8-cell embryo with a whole 8-cell embryo. As expected, the overall contribution of 4-cell embryos was lower than that of 8-cell embryos and similar to that of half 8-cell stage embryos. In the 4-cell[harr ]8-cell chimaeras the 4-cell stage embryos did not contribute more to the trophectoderm than the ICM derivatives. Thus, differences between 4-cell and 8-cell embryos cannot explain the restricted tissue distribution of tetraploid cells previously reported for 4-cell tetraploid[harr ]8-cell diploid chimaeras. It is suggested that cells from the more advanced embryo are more likely to contribute to the ICM but, for technical reasons, are prevented from doing so in simple aggregates of equal numbers of whole 4-cell and whole 8-cell stage embryos.

Vitrification of biopsied mouse embryos

2005 ◽  
Vol 53 (1) ◽  
pp. 103-112 ◽  
Author(s):  
B. Baranyai ◽  
Sz. Bodó ◽  
◽  
Keyword(s):  
Solid Surface ◽  
Birth Rate ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
The Novel ◽  
Cell Stage ◽  
Cryopreservation Protocol

Solid surface vitrification (SSV) was compared with in-straw vitrification for cryopreservation of biopsied mouse embryos. Eight-cell stage embryos were zona drilled and one blastomere was removed. Developed morulae or blastocysts were vitrified in microdrop (35% EG + 5% PVP + 0.4 M trehalose) or in straw (7.0 M EG + 0.5 M sucrose). Following recovery, embryos were cultivated in vi tro or transferred into recipients. Cryopreservation had an effect not only on the survival of biopsied embryos but also on their subsequent development in vitro. Cryosurvival of biopsied morulae vitrified in straw was significantly inferior to SSV. The post-warm development of biopsied and non-biopsied morulae was delayed on Day 3.5 and 4.5 in both vitrification groups. A delay in development was observed on Day 5.5 among vitrified non-biopsied blastocysts. The percentage of pups born from biopsied morulae or blastocysts following cryopreservation did not differ from that of the control. No significant differences could be detected between methods within and between embryonic stages in terms of birth rate. The birth rate of biopsied embryos vitrified in straw was significantly lower compared to the non-biopsied embryos. The novel cryopreservation protocol of SSV proved to be effective for cryopreservation of morula- and blastocyst-stage biopsied embryos.

Distribution of introduced human mitochondrial DNA in early stage mouse embryos

2020 ◽  
Vol 20 (2) ◽  
pp. 69-78
Author(s):  
Maria E. Kustova ◽  
Vasilina A. Sokolova ◽  
Oksana V. Kidgotko ◽  
Mikhail G. Bass ◽  
Faina M. Zakharova ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Early Stage ◽  
Proper Time ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Hormonal Stimulation ◽  
Human Mitochondrial Dna ◽  
Cell Embryo ◽  
Mitochondrial Suspension ◽  
Species Specific

Objective. The aim of study was the analysis of human mitochondrial DNA (mtDNA) distribution among murine blastomeres in the embryos developing after an injection of human mitochondria suspension at the stage of one or two cells is presented. Material and methods. Mice CBA/C57Black from Rappolovo aged three weeks were used. Zygotes were obtained upon hormonal stimulation of animals and mated with males. 310 pL of mitochondrial suspension from HepG2 cells was injected into a zygote or one blastomere of a two-cell embryo. Zygotes or two-cell embryos cultured in M3 medium drops covered with mineral oil in Petri dishes. Upon reaching the two-, four- or eight-cell stage the cultured embryos were separated into blastomeres. The latter were lysed and the total DNA was isolated. Human mtDNA was detected by PCR using species-specific primers. Results. The development of 2848 mouse embryos was monitored. In 520 embryos that achieved the stage of 2, 4, 8 in proper time the presence of human mtDNA was assayed in each blastomere. Along with murine mtDNA all embryos contained human mitochondrial genome, which is an evidence of artificially modelled heteroplasmy. Not every blastomere of transmitochondrial embryos contained foreign (human) mtDNA. Mathematical elaboration evidenced an uneven distribution of human mtDNA in cytoplasm within the time elapsed between the injection of human mitochondria and the subsequent splitting of the embryo. Conclusion. The results obtained confirm our previous notion of the presence of 1011 segregation units of human mtDNA in the total amount of mitochondria (about 5 ∙ 102) injected into an embryo.

Muscle cell differentiation in ascidian embryos analysed with a tissue-specific monoclonal antibody

Development ◽  
1987 ◽  
Vol 99 (2) ◽  
pp. 163-171
Author(s):  
T. Nishikata ◽  
I. Mita-Miyazawa ◽  
T. Deno ◽  
N. Satoh
Keyword(s):  
Monoclonal Antibody ◽  
Cell Differentiation ◽  
Muscle Cell ◽  
Antigen Expression ◽  
Relative Molecular Mass ◽  
Specific Monoclonal Antibody ◽  
Cell Stage ◽  
Muscle Cell Differentiation ◽  
Cell Embryo ◽  
Ascidian Embryos

Utilizing a muscle-specific monoclonal antibody (Mu-2) as a probe, we analysed developmental mechanisms involved in muscle cell differentiation in ascidian embryos. The antigen recognized by Mu-2 was a single polypeptide with a relative molecular mass of about 220 X 10(3). It first appeared at the early tailbud stage and continued to be expressed until the swimming larva stage. There were distinct and separate puromycin and actinomycin D sensitivity periods during the occurrence of the antigen, suggesting the new synthesis of the polypeptide by developing muscle cells. Embryos that had been permanently arrested with aphidicolin in the early cleavage stages up to the 32-cell stage did not express the antigen. DNA replications may be required for the antigen expression. Embryos that had been arrested with cytochalasin B in the 8-cell and later stages developed the antigen, and the number and position of the arrested blastomeres exhibiting the differentiation marker almost corresponded to those of the B4.1-line muscle lineage. Furthermore, in quarter embryos developed from each blastomere pair isolated from the 8-cell embryo, all the B4.1 as well as a part of b4.2 partial embryos expressed the antigen, while the a4.2 and A4.1 partial embryos did not show the antigen expression. These results may provide further support for the existence of cytoplasmic determinants for muscle cell differentiation in this mosaic egg.

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