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.

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.

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 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.

scholarly journals MTG16 (CBFA2T3) represses E protein-dependent transcription to regulate colonic secretory cell differentiation, epithelial regeneration, and tumorigenesis

2021 ◽  
Author(s):  
Rachel E. Brown ◽  
Justin Jacobse ◽  
Shruti A. Anant ◽  
Koral M. Blunt ◽  
Bob Chen ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Mouse Model ◽  
Cell Fate ◽  
Protein Interactions ◽  
Protein Function ◽  
Colonic Epithelium ◽  
E Proteins ◽  
Cell Fate Decisions ◽  
Epithelial Regeneration ◽  
E Protein

Aberrant epithelial differentiation and regeneration pathways contribute to colon pathologies including inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). MTG16 (also known as CBFA2T3) is a transcriptional corepressor expressed in the colonic epithelium. MTG16 interaction partners include E box-binding basic helix-loop-helix transcription factors (E proteins). MTG16-deficient mice exhibit worse colitis and increased tumor burden in inflammatory carcinogenesis. In this study, we sought to understand the role of MTG16 in colonic epithelial homeostasis and the mechanisms by which MTG16 protects the epithelium in colitis and CAC. We demonstrated that MTG16 deficiency enabled enteroendocrine cell differentiation from secretory precursor cells at the expense of goblet cells. Transcriptomic analysis implicated dysregulated E protein function in MTG16-deficient colon crypts. Using a novel mouse model with a point mutation that disrupts MTG16:E protein complex formation (Mtg16P209T), we established that enteroendocrine:goblet cell balance was dependent on MTG16:E protein interactions and that the shift in lineage allocation was associated with enhanced expression of Neurog3, the central driver of enteroendocrine lineage specification. Furthermore, Mtg16 was upregulated in the previously described Ascl2+, de-differentiating cells that replenish the stem cell compartment in response to colon injury. Mtg16 expression was also increased in dextran sulfate sodium (DSS)-treated mouse colon crypts and in IBD patients compared to unaffected controls. We determined that the effects of MTG16 in regeneration are also dependent on its repression of E proteins, as the colonic epithelium failed to regenerate following DSS-induced injury in our novel mutant mouse model. Finally, we revealed that uncoupling MTG16:E protein interactions contributes to the enhanced tumorigenicity in Mtg16-/- colon in the azoxymethane(AOM)/DSS-induced model of CAC. Collectively, our results demonstrate that MTG16, via its repression of E protein targets, is a key regulator of cell fate decisions during colonic differentiation and regeneration.

scholarly journals Pannexin 1 influences lineage specification of human iPSCs

2021 ◽  
Author(s):  
Rebecca J. Noort ◽  
Grace A. Christopher ◽  
Jessica L. Esseltine
Keyword(s):  
Cell Fate ◽  
Cell Communication ◽  
Cell Lineage ◽  
The Body ◽  
Human Embryos ◽  
Lineage Specification ◽  
Cell Stage ◽  
Cell Fate Decisions ◽  
Pannexin 1 ◽  
Human Ipscs

AbstractEvery single cell in the body communicates with nearby cells to locally organize activities with their neighbors and dysfunctional cell-cell communication can be detrimental during cell lineage commitment, tissue patterning and organ development. Pannexin channels (PANX1, PANX2, PANX3) facilitate purinergic paracrine signaling through the passage of messenger molecules out of cells. PANX1 is widely expressed throughout the body and has recently been identified in human oocytes as well as 2 and 4-cell stage human embryos. Given its abundance across multiple adult tissues and its expression at the earliest stages of human development, we sought to understand whether PANX1 impacts human induced pluripotent stem cells (iPSCs) or plays a role in cell fate decisions. Western blot, immunofluorescence and flow cytometry reveal that PANX1 is expressed in iPSCs as well as all three germ lineages derived from these cells: ectoderm, endoderm, and mesoderm. PANX1 demonstrates differential glycosylation patterns and subcellular localization across the germ lineages. Using CRISPR-Cas9 gene ablation, we find that loss of PANX1 has no obvious impact on iPSC morphology, survival, or pluripotency gene expression. However, PANX1 knockout iPSCs exhibit apparent lineage specification bias during 2-dimensional and 3-dimensional spontaneous differentiation into the three germ lineages. Indeed, loss of PANX1 significantly decreases the proportion of ectodermal cells within spontaneously differentiated cultures, while endodermal and mesodermal representation is increased in PANX1 knockout cells. Importantly, PANX1 knockout iPSCs are fully capable of differentiating toward each specific lineage when exposed to the appropriate external signaling pressures, suggesting that although PANX1 influences germ lineage specification, it is not essential to this process.Graphical abstract

scholarly journals Helios represses megakaryocyte priming in hematopoietic stem and progenitor cells

2021 ◽  
Vol 218 (10) ◽  
Author(s):  
Giovanni Cova ◽  
Chiara Taroni ◽  
Marie-Céline Deau ◽  
Qi Cai ◽  
Vincent Mittelheisser ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Fate ◽  
Transcriptional Activators ◽  
Cell Stage ◽  
Hematopoietic Stem ◽  
Chromatin Compaction ◽  
Cell Fate Decisions ◽  
Quiescent Cells ◽  
Stem And Progenitor Cells ◽  
Expression Program

Our understanding of cell fate decisions in hematopoietic stem cells is incomplete. Here, we show that the transcription factor Helios is highly expressed in murine hematopoietic stem and progenitor cells (HSPCs), where it is required to suppress the separation of the platelet/megakaryocyte lineage from the HSPC pool. Helios acts mainly in quiescent cells, where it directly represses the megakaryocyte gene expression program in cells as early as the stem cell stage. Helios binding promotes chromatin compaction, notably at the regulatory regions of platelet-specific genes recognized by the Gata2 and Runx1 transcriptional activators, implicated in megakaryocyte priming. Helios null HSPCs are biased toward the megakaryocyte lineage at the expense of the lymphoid and partially resemble cells of aging animals. We propose that Helios acts as a guardian of HSPC pluripotency by continuously repressing the megakaryocyte fate, which in turn allows downstream lymphoid priming to take place. These results highlight the importance of negative and positive priming events in lineage commitment.

scholarly journals ADAM10 is essential for Notch2-dependent marginal zone B cell development and CD23 cleavage in vivo

2010 ◽  
Vol 207 (3) ◽  
pp. 623-635 ◽  
Author(s):  
David R. Gibb ◽  
Mohey El Shikh ◽  
Dae-Joong Kang ◽  
Warren J. Rowe ◽  
Rania El Sayed ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Fate ◽  
Marginal Zone ◽  
Cell Development ◽  
B Cell Development ◽  
Mouse Embryos ◽  
Cell Fate Decisions ◽  
Important Regulator ◽  
A Disintegrin And Metalloproteinase

The proteolytic activity of a disintegrin and metalloproteinase 10 (ADAM10) regulates cell-fate decisions in Drosophila and mouse embryos. However, in utero lethality of ADAM10−/− mice has prevented examination of ADAM10 cleavage events in lymphocytes. To investigate their role in B cell development, we generated B cell–specific ADAM10 knockout mice. Intriguingly, deletion of ADAM10 prevented development of the entire marginal zone B cell (MZB) lineage. Additionally, cleavage of the low affinity IgE receptor, CD23, was profoundly impaired, but subsequent experiments demonstrated that ADAM10 regulates CD23 cleavage and MZB development by independent mechanisms. Development of MZBs is dependent on Notch2 signaling, which requires proteolysis of the Notch2 receptor by a previously unidentified proteinase. Further experiments revealed that Notch2 signaling is severely impaired in ADAM10-null B cells. Thus, ADAM10 critically regulates MZB development by initiating Notch2 signaling. This study identifies ADAM10 as the in vivo CD23 sheddase and an important regulator of B cell development. Moreover, it has important implications for the treatment of numerous CD23- and Notch-mediated pathologies, ranging from allergy to cancer.

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 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.

scholarly journals Primed Track, high-fidelity lineage tracing in mouse pre-implantation embryos using primed conversion of photoconvertible proteins

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Maaike Welling ◽  
Manuel Alexander Mohr ◽  
Aaron Ponti ◽  
Lluc Rullan Sabater ◽  
Andrea Boni ◽  
...  
Keyword(s):  
Cell Fate ◽  
Cell Tracking ◽  
Fluorescent Proteins ◽  
Time Windows ◽  
Lineage Tracing ◽  
Fluorescence Labeling ◽  
High Fidelity ◽  
Imaging Data ◽  
Cell Stage ◽  
Cell Fate Decisions

Accurate lineage reconstruction of mammalian pre-implantation development is essential for inferring the earliest cell fate decisions. Lineage tracing using global fluorescence labeling techniques is complicated by increasing cell density and rapid embryo rotation, which hampers automatic alignment and accurate cell tracking of obtained four-dimensional imaging data sets. Here, we exploit the advantageous properties of primed convertible fluorescent proteins (pr-pcFPs) to simultaneously visualize the global green and the photoconverted red population in order to minimize tracking uncertainties over prolonged time windows. Confined primed conversion of H2B-pr-mEosFP-labeled nuclei combined with light-sheet imaging greatly facilitates segmentation, classification, and tracking of individual nuclei from the 4-cell stage up to the blastocyst. Using green and red labels as fiducial markers, we computationally correct for rotational and translational drift, reduce overall data size, and accomplish high-fidelity lineage tracing even for increased imaging time intervals – addressing major concerns in the field of volumetric embryo imaging.

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