Position- and Hippo signaling-dependent plasticity during lineage segregation in the early mouse embryo

Pósfai, Eszter; Petropoulos, Sophie; Barros, Flávia Regina Oliveira de; Schell, John P.; Jurišica, Igor; Sandberg, Rickard; Lanner, Fredrik; Rossant, Janet

doi:10.7554/elife.22906

Cited by 146 publications

(186 citation statements)

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“…Cytoplasmic microinjection of zygotes and 2-cell stage embryos were performed as described previously 10 . Briefly, Cas9 mRNA (100ng/μl) or Cas9-mSA mRNA (75ng/μl), sgRNA (50ng/μl) and donor plasmids (30ng/μl) or BIO-PCR donors (20ng/μl) were microinjected in nuclease-free injection buffer (10mM Tris-HCl, pH 7.4, 0.25mM EDTA).…”

Section: Cytoplasmic Microinjection Of Zygote and 2-cell Stage Embryosmentioning

confidence: 99%

“…Fluorescent reporter mouse lines, which allow for direct visualization and live imaging of molecular and cellular dynamics during developmental, regenerative and pathological processes are among the most commonly generated models using large fragment knock-in. The preimplantation mouse embryo is an ideal model to study basic developmental mechanisms by live imaging 10 . Two consecutive crucial cell fate decisions separate embryonic and extraembryonic lineages and define three cell types in a 4-day time frame 10 .…”

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confidence: 99%

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confidence: 99%

“…The preimplantation mouse embryo is an ideal model to study basic developmental mechanisms by live imaging 10 . Two consecutive crucial cell fate decisions separate embryonic and extraembryonic lineages and define three cell types in a 4-day time frame 10 . Moreover, because of the highly accessible nature of preimplantation embryos, they serve as excellent models to develop and apply cutting edge imaging and gene manipulation techniques such as single molecule tracking 11 , optogenetics and induced protein degradation systems 12,13 .…”

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confidence: 99%

“…We selected most "classical" lineage markers covering all three cell types of the blastocyst. These included epibalst (EPI) (Sox2, Nanog, Oct4 and Esrrb), primitive endoderm (PE) (Gata6, Gata4, Sox17, Fgfr2 and Pdgfra) and trophectoderm (TE) (Cdx2, Eomes and Gata3) markers 10 (Fig. 1c).…”

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

Pósfai

Rossant

2017

Preprint

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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 biotinstreptavidin 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 . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/204339 doi: bioRxiv preprint first posted online Oct. 16, 2017; 2 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 development of endonuclease-mediated genome editing technologies, in particular CRISPR/Cas9, has revolutionized mouse genetics by opening up the possibility to bypass the long and labor intensive embryonic stem cell-based methods and achieve precise genome editing directly in zygote stage embryos 1 .CRISPR/Cas9-mediated genome editing has shown consistently high efficiency in generating indels, point mutations and small insertions in zygotes 2,3 , largely making use of the non-homologous end-joining (NHEJ) and homology directed repair (HDR) pathways. However, the efficiency of precise introduction of large fragments by homologous recombination (HR) is still not ideal 4,5 . Previous reports have suggested methods to increase large fragment knock-in efficiency in zygotes, by Cas9-RNP injection 6 , with templates activating micro-homology mediated end joining or long-homology mediated end joining pathways 4,7 , with long singlestranded DNA templates or by chemically manipulating DNA repair pathways 8,9 .However, only a limited number of successfully targeted loci have been reported to . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/204339 doi: bioRxiv preprint first posted online Oct. 16, 2017; 3 date 4 . Some methods produce imprecise junctions at editing sites 7 , while others are limited by practi...

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Section: Cytoplasmic Microinjection Of Zygote and 2-cell Stage Embryosmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Pósfai

Rossant

2017

Preprint

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Localization of YAP activity in developing skeletal rudiments is responsive to mechanical stimulation

Shea

Rolfe

McNeill

et al. 2019

Developmental Dynamics

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Background Normal skeletal development, in particular ossification, joint formation and shape features of condyles, depends on appropriate mechanical input from embryonic movement but it is unknown how such physical stimuli are transduced to alter gene regulation. Hippo/Yes‐Associated Protein (YAP) signalling has been shown to respond to the physical environment of the cell and here we specifically investigate the YAP effector of the pathway as a potential mechanoresponsive mediator in the developing limb skeleton. Results We show spatial localization of YAP protein and of pathway target gene expression within developing skeletal rudiments where predicted biophysical stimuli patterns and shape are affected in immobilization models, coincident with the period of sensitivity to movement, but not coincident with the expression of the Hippo receptor Fat4. Furthermore, we show that under reduced mechanical stimulation, in immobile, muscle‐less mouse embryos, this spatial localization is lost. In culture blocking YAP reduces chondrogenesis but the effect differs depending on the timing and/or level of YAP reduction. Conclusions These findings implicate YAP signalling, independent of Fat4, in the transduction of mechanical signals during key stages of skeletal patterning in the developing limb, in particular endochondral ossification and shape emergence, as well as patterning of tissues at the developing synovial joint.

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CDX2 downregulation in mouse mural trophectoderm during peri‐implantation is heteronomous, dependent on the YAP‐TEAD pathway and controlled by estrogen‐induced factors

Suzuki

Okura

Nagakura

et al. 2022

Reprod Medicine & Biology

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Purpose To investigate the transition of CDX2 expression patterns in mouse trophectoderm (TE) and its regulatory mechanisms during implantation. Methods Mouse E3.5–4.5 blastocysts were used to immunostain CDX2, YAP, TEAD4, and ESRRB. Endogenous estrogen signaling was perturbed by administrating estrogen receptor antagonist ICI 182,780 or ovariectomy followed by administration of progesterone and β‐estradiol to elucidate the relationship between the transition of CDX2 expression patterns and ovarian estrogen‐dependent change in the uterine environment. Results CDX2 expression was gradually downregulated in the mural TE from E4.0 in vivo , whereas CDX2 downregulation was not observed in blastocysts cultured in KSOM. Fetal bovine serum (FBS) supplementation in KSOM induced CDX2 downregulation independently of blastocyst attachment to dishes. CDX2 downregulation in the mural TE was repressed by administration of ICI 182,780 or by ovariectomy, and administration of β‐estradiol into ovariectomized mice retriggered CDX2 downregulation. Furthermore, Cdx2 expression in the mural TE might be controlled by the YAP‐TEAD pathway. Conclusions CDX2 downregulation was induced heteronomously in the mural TE from E4.0 by uterus‐derived factors, the secretion of which was stimulated by ovarian estrogen.

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Position- and Hippo signaling-dependent plasticity during lineage segregation in the early mouse embryo

Cited by 146 publications

References 62 publications

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

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

Localization of YAP activity in developing skeletal rudiments is responsive to mechanical stimulation

CDX2 downregulation in mouse mural trophectoderm during peri‐implantation is heteronomous, dependent on the YAP‐TEAD pathway and controlled by estrogen‐induced factors

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