Noninvasive visualization of molecular events in the mammalian zygote

Yamagata, Kazuya; Yamazaki, Taiga; Yamashita, Misuzu; Hara, Yuki; Ogonuki, Narumi; Ogura, Atsuo

doi:10.1002/gene.20158

Cited by 89 publications

(75 citation statements)

References 29 publications

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“…For the expression of Bysl-Venus in embryos, the EGFP cDNA in the pcDNA3.1EGFP-poly(A83) vector (70) was replaced by the cDNA encoding Bysl-Venus. After linearization of the expression vector, in vitro mRNA transcription was performed using the RiboMAX Large Scale RNA Production Systems-T7 kit (Promega) in the presence of m 7 G(5Ј)ppp(5Ј)G RNA capping (44) were cultured in the absence of feeder cells in Dulbecco's modified Eagle medium supplemented with 15% fetal calf serum, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 0.1 mM nonessential amino acids, 50 U/ml penicillin-streptomycin, 1,000 U/ml of leukemia inhibitory factor (LIF), 100 g/ml G418, and 10 g/ml zeocin on gelatin-coated dishes.…”

Section: Methodsmentioning

confidence: 99%

Crucial Role of Bysl in Mammalian Preimplantation Development as an Integral Factor for 40S Ribosome Biogenesis

Adachi

Soeta-Saneyoshi

Sagara

et al. 2007

Molecular and Cellular Biology

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Blastocyst formation during mammalian preimplantation development is a unique developmental process that involves lineage segregation between the inner cell mass and the trophectoderm. To elucidate the molecular mechanisms underlying blastocyst formation, we have functionally screened a subset of preimplantation embryo-associated transcripts by using small interfering RNA (siRNA) and identified Bysl (bystin-like) as an essential gene for this process. The development of embryos injected with Bysl siRNA was arrested just prior to blastocyst formation, resulting in a defect in trophectoderm differentiation. Silencing of Bysl by using an episomal short hairpin RNA expression vector inhibited proliferation of embryonic stem cells. Exogenously expressed Bysl tagged with a fluorescent protein was concentrated in the nucleolus with a diffuse nucleoplasmic distribution. Furthermore, the loss of Bysl function by using RNA interference or dominant negative mutants caused defects in 40S ribosomal subunit biogenesis. These findings provide evidence for a crucial role of Bysl as an integral factor for ribosome biogenesis and suggest a critical dependence of blastocyst formation on active translation machinery.Blastocyst formation is the first differentiation process during mammalian embryonic development, leading to the segregation of two distinct cell lineages: the inner cell mass (ICM) and the trophectoderm (TE) (27). Prior to blastocyst formation, intercellular junctions are established during compaction and cellular polarity is developed. The subsequent rounds of asymmetric divisions give rise to spatially segregated outer and inner cells. The outer layer of cells adopts a polarized epithelial structure differentiating into the TE, which then contributes to extraembryonic tissues, including the placenta. The apolar ICM is the founder cell population of the embryo proper. The ICM and its successor, the epiblast, comprise transient pluripotent stem cell populations. After implantation, the cells begin to differentiate into specific cell types that will form the three primary germ layers. Embryonic stem (ES) cells, which are derived from the ICM or epiblast, self-renew and maintain pluripotent differentiation capacity in culture. Although the regulatory networks that are responsible for the maintenance of ES cell identity have been extensively investigated, molecular analysis of preimplantation development is quite limited due to the scarcity of materials and experimental approaches.With the advent of the genome projects, global gene expression profiles of various tissues and developmental stages have been generated from cDNA libraries and microarray analyses. These studies have provided an effective way to identify genes that are preferentially expressed in specific cell types or tissues, especially in early embryos from which only small amounts of material can be obtained. In addition, gene silencing by RNA interference (RNAi) has been established as a method for reverse genetic analysis of gene function in mammalian cell...

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Section: Methodsmentioning

confidence: 99%

Crucial Role of Bysl in Mammalian Preimplantation Development as an Integral Factor for 40S Ribosome Biogenesis

Adachi

Soeta-Saneyoshi

Sagara

et al. 2007

Molecular and Cellular Biology

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“…The plasmids of mRNA for EGFP-α-tubulin [14] and histone H2B-mRFP1 [24] inserted in pcDNA3.1poly(A83) vector were as constructed previously. EGFP-β-actin was generated by fusion of the EGFP with the N-terminal of full-length cDNA encoding mouse β-actin.…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…When such resulting embryos are immunostained, some abnormalities including cytogenetic and epigenetic aberrations can be detected, but it is impossible to ascertain the influence of such abnormalities on subsequent development. However, the live-cell imaging approach allows us to link a specific phenomenon observed directly at a certain point to each embryo's developmental capacity by culturing and transplanting it to a recipient pseudopregnant mother.We have developed a live-cell imaging technique for mammalian preimplantation embryos [14] in which embryos are microinjected with mRNA encoding fluorescent proteins and then observed by time-lapse microscopy. Using this technique, we analyzed global changes in DNA methylation status during preimplantation development in normal and cloned embryos [15,9].…”

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Long-Term, Six-Dimensional Live-Cell Imaging for the Mouse Preimplantation Embryo That Does Not Affect Full-Term Development

Yamagata

Suetsugu

Wakayama

2009

Journal of Reproduction and Development

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Abstract. Mammalian preimplantation embryonic development is achieved by tightly coordinated regulation of a great variety of temporal and spatial changes. Therefore, it would be valuable to analyze these events three-dimensionally and dynamically. We have previously developed a live-cell imaging method based on the expression of fluorescent proteins, using mRNA injection and time-lapse florescence microscopy. However, with conventional fluorescent microscopy, three-dimensional images could not be obtained due to the thickness of the embryos and the optical problem in which 'out-of focus blur' cannot be eliminated. Moreover, as the repeated exposure of intense excitation light to the cell yields phototoxicity, long-term observation was detrimental to embryonic development. Here, we improved our imaging system to enable six-dimensional live-cell imaging of mouse preimplantation embryos (x, y and z axes, time-lapse, multicolor and multisample). Importantly, by improving the imaging devices and optimizing the conditions for imaging, such as intensity of excitation and time intervals for image acquisition, the procedure itself was not detrimental to full-term development, although it is a prolonged imaging process. For example, live pups were obtained from embryos to which two different wavelengths of excitation (488 and 561 nm) were applied at 7.5-min intervals for about 70 h, and 51 images were acquired in the z axis at each time point; thus, a total of 56,814 fluorescent images were taken. All the pups were healthy, reproductively normal and not transgenic. Thus, this live-cell imaging technology is safe for full-term mouse development. This offers a novel approach for developmental and reproductive research in that it enables both retrospective and prospective analyses of development. It might also be applicable to assessment of embryo quality in fields such as human reproductive technology and production animal research. Key words: Embryo assessment, Full-term development, Live-cell imaging, Preimplantation embryo, Spinning-disk confocal microscopy (J. Reprod. Dev. 55: [343][344][345][346][347][348][349][350] 2009) ammalian preimplantation development is a process in which the union of two highly specialized gametes endows the resulting zygote with the potential to form undifferentiated cell types. This is followed by cell proliferation and the beginning of differentiation during the brief period leading up to implantation. In these processes, a number of cellular components and structures are regulated spatially and temporally, as seen in repeated cell division, cell cycle progression and genomic reprogramming via global epigenetic modifications [1]. Hence, many researchers have been attracted to these fundamental biological processes. However, the numbers of oocytes and embryos that can be collected are very limited, especially in mammals. Therefore, analysis of molecular mechanisms is hampered because of difficulties in conducting biochemical analyses on sufficient materials. Also, immunostaining methods ...

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“…After immunostaining for CDX2, blastocysts were incubated in PBS containing 1 mg/ml PI. Serial confocal images were taken using fluorescence confocal microscopy (Yamagata et al 2005), and three-dimensional images of blastocysts were reconstructed using MetaMorph software (Universal Imaging Co., Downingtown, PA, USA). More than ten blastocysts were stained and the total (PI-positive) and TE (CDX2-positive) cell numbers were determined.…”

Section: Assessment Of Cell Numbers Of Blastocystsmentioning

confidence: 99%

Generation of mice derived from embryonic stem cells using blastocysts of different developmental ages

Ohta¹,

Sakaide²,

Wakayama³

2008

Reproduction

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We previously showed that increasing the cell number of host tetraploid (4n) embryos by aggregating multiple 4n embryos at two to fourcell stages can improve the birthrate of mice from embryonic stem cells (ES mice). In the present study, we assessed whether in vitro aged blastocysts (e.g., E4.5 or E5.5), where their cell number also increased with development, can be used as hosts for generating ES mice. As expected, the cell number of in vitro aged 4n blastocysts increased with development, i.e., 26.5G2.4, 49.6G8.4, and 84.9G20.9 cells for E3.5, E4.5, and E5.5 respectively. Three independent ES cell lines were injected into 4n aged blastocysts, and their developmental ability was compared with that of E3.5 4n blastocysts commonly used for this procedure. We found that the birthrate of ES mice derived from E4.5 blastocysts were comparable with those of mice generated from E3.5 blastocysts. On the other hand, the birthrates decreased when E5.5 blastocysts were used. These results suggest that not only the cell number but also developmental age is important for producing ES mice. We also discuss a comparison of the present findings with those of our previous study, where ES mice were generated using an aggregation method employing the same ES cell lines.

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Noninvasive visualization of molecular events in the mammalian zygote

Cited by 89 publications

References 29 publications

Crucial Role of Bysl in Mammalian Preimplantation Development as an Integral Factor for 40S Ribosome Biogenesis

Crucial Role of Bysl in Mammalian Preimplantation Development as an Integral Factor for 40S Ribosome Biogenesis

Long-Term, Six-Dimensional Live-Cell Imaging for the Mouse Preimplantation Embryo That Does Not Affect Full-Term Development

Generation of mice derived from embryonic stem cells using blastocysts of different developmental ages

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