Gastrulation events in the prestreak pig embryo: Ultrastructure and cell markers

Fléchon, J.‐E.; Degrouard, Jéril; Fléchon, B.

doi:10.1002/gene.10244

Cited by 59 publications

(45 citation statements)

References 53 publications

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“…During the immediate post-hatching period, however, Oct-3/4 expression becomes restricted to the ICM and epiblast [28,[50][51][52]. In the present study, localization of EGFP expression in blastocysts developed from embryos reconstituted with pOEIN-MPFF-#4 cells was not only confined to the ICM but was even seen in the TE.…”

Section: Discussionsupporting

confidence: 45%

Development of a Noninvasive Monitoring System for Evaluation of Oct-3/4 Promoter Status in Miniature Pig Somatic Cell Nuclear Transfer Embryos

Miyoshi

Mori

Mizobe

et al. 2009

Journal of Reproduction and Development

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Abstract. The present study was carried out to develop a noninvasive monitoring system for evaluation of Oct-3/4 promoter gene status in miniature pig somatic cell nuclear transfer (SCNT) embryos during in vitro development. Miniature pig fetal fibroblasts (MPFFs) were transfected with a gene construct consisting of two expression units, a mouse Oct-3/4 promoter-driven enhanced green fluorescent protein (EGFP) gene (EGFP expression only detected in Oct-3/4-expressing cells) and a neomycin resistance gene. After neomycin selection, MPFFs that did not express EGFP were fused with enucleated pig oocytes, cultured in vitro and assessed for EGFP expression. EGFP expression was detectable in all morulae (at 4-6 days of culture) and 50.0% of blastocysts (at 5-6 days of culture), whereas none of the 1-cell to 16-cell embryos at 1-5 days of culture expressed EGFP. On the other hand, EGFP expression was not maintained in all blastocysts at 7 days of culture. The reactivity with anti-Oct-3/4 antibodies also peaked from the morula to blastocyst stages at 5 days of culture. The results showed that reactivation of the Oct-3/4 promoter gene of donor nuclei occurs in the morula to blastocyst stages at 4-6 days after SCNT and that this noninvasive monitoring system using Oct-3/4 promoter-driven EGFP gene would be useful for evaluation of the reprogramming status of donor nuclei. Key words: Enhanced green fluorescent protein, In vitro development, Nuclear transfer, Oct-3/4 promoter, Reprogramming (J. Reprod. Dev. 55: [661][662][663][664][665][666][667][668][669] 2009) uccessful production of cloned animals using somatic cell nuclear transfer (SCNT) has been reported in several mammalian species [1][2][3][4][5][6][7][8][9][10][11][12]. However, the low cloning efficiency associated with development of SCNT embryos to offspring remains the major obstacle to widespread use of this technology in a number of animal science and biomedical applications. A method for evaluating the developmental ability of SCNT embryos before being transferred into recipient females should be explored to optimize the procedures of SCNT and overcome the low cloning efficiency. Evaluation of the in vitro developmental ability of an SCNT embryo to the blastocyst stage would be considered one of the methods for predicting its in vivo development, but it has been found that this does not often assure successful development to term of SCNT embryos [13]. After SCNT, the gene expression pattern in somatic cells is reprogrammed to mimic that in preimplantation embryos [14]. However, there is a difference in gene expression pattern between SCNT embryos and in vitro-fertilized embryos, suggesting that the low efficiency of SCNT is associated with incomplete reprogramming in the donor nuclei transferred into recipient oocytes [15]. Therefore, evaluation of the reprogramming level in the donor nuclei appears to be most essential for predicting the in vivo developmental ability of SCNT embryos.Oct-3/4 is a transcription factor of the Pit-Oct-Unc family [16,17], and...

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Section: Discussionsupporting

confidence: 45%

Development of a Noninvasive Monitoring System for Evaluation of Oct-3/4 Promoter Status in Miniature Pig Somatic Cell Nuclear Transfer Embryos

Miyoshi

Mori

Mizobe

et al. 2009

Journal of Reproduction and Development

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“…Stem cells express a core group of genes that are critical for maintaining pluripotency [ 28 ]. Our analysis of pure epiblasts shows that these cells express the core genes NANOG , OCT4 , and SOX2 and the epiblast marker NODAL .…”

Section: Discussionmentioning

confidence: 84%

Pig Epiblast Stem Cells Depend on Activin/Nodal Signaling for Pluripotency and Self-Renewal

Alberio

Croxall

Allegrucci

2010

Stem Cells and Development

107

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Activin/Nodal signaling is required for maintaining pluripotency and self-renewal of mouse epiblast stem cells and human embryonic stem cells (hESC). In this study, we investigated whether this signaling mechanism is also operative in cultured epiblasts derived from Days 10.5-12 pig embryos. Pig epiblast stem cell lines (pEpiSC) were established on mouse feeder layers and medium supplemented with basic fi broblast growth factor (bFGF). pEpiSC express the core pluripotency factors OCT4 (or POU5F1 ), NANOG , SOX2 , and NODAL , but they do not express REX1 or alkaline phosphatase activity. Blocking leukemia inhibitory factor (LIF)/JAK/STAT3 pathway by adding the specifi c JAK I inhibitor 420099 and an anti-LIF antibody over 3 passages did not affect pluripotency of pEpiSC. In contrast, cells grown with the Alk-5 inhibitor SB431542, which blocks Activin/Nodal pathway, differentiated readily toward the neural lineage. pEpiSC are pluripotent, as established by their differentiation potential to ectoderm, mesoderm, and endoderm. These cells can be induced to differentiate toward trophectoderm and to germ cell precursors in response to bone morphogenetic protein 4 (BMP-4). In conclusion, our study demonstrates that pig epiblasts express the core pluripotency genes and that the capacity for maintaining self-renewal in pEpiSC depends on Activin/Nodal signaling. This study provides further evidence that maintenance of pluripotency via Activin/Nodal signal is conserved in mammals. Introduction Mouse embryonic stem cells (mESC), conventionally derived from the inner cell mass (ICM) of preimplantation blastocysts, are pluripotent and can self-renew indefinitely in culture. mESC depend on the cytokines leukemia inhibitory factor (LIF) and bone morphogenetic protein 4 (BMP-4) to maintain the undifferentiated state [ 1 , 2 ]. Human embryonic stem cells (hESC) are also derived from blastocysts; however, these cells depend on basic fi broblast growth factor (bFGF) and Activin A for pluripotency and selfrenewal [ 3 , 4 ]. Interestingly, pluripotent cells derived from mouse epiblasts, a part of the ICM, also require bFGF and Activin A for pluripotency and self-renewal [ 5 , 6 ]. hESC share multiple features with mouse epiblast stem cells (mEpiSC), such as growing as fl at and compact colonies [5][6][7], they respond to BMP-4 by differentiating to trophectoderm (TE) [ 8 ] and germ cell progenitors [ 9 ], and they do not require LIF/ JAK/STAT3 signaling for self-renewal [ 4 , 10 ]. The functional and phenotypic similarities between these cell types suggest a developmental relationship [ 5 , 6 ], and demonstrate that there are at least 2 signaling mechanisms involved in capturing pluripotency in vitro. Recent reports show that bFGF and Activin A are also necessary for maintaining rabbit ESC [ 11 , 12 ], indicating that this signaling pathway may be a conserved mechanism of pluripotency in mammals.Here we tested this hypothesis in pigs, a representative of ungulates. Attempts to derive stem cells from pig embryos have traditionally reli...

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“…During embryonic development in the mouse, vimentin is first expressed at E6.5-E7.0, and it is used as a marker for type 1 EMT during gastrulation (80,81). However, because adult epithelial cells transiently express vimentin in response to various insults (82), vimentin should not be considered a marker of type 2 EMT in the setting of fibrosis.…”

Section: Cell-surface Markers Of Emt a Change In Expression Of E-cadmentioning

confidence: 99%

Biomarkers for epithelial-mesenchymal transitions

Zeisberg¹,

Neilson²

2009

J. Clin. Invest.

1,998

1,895

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Somatic cells that change from one mature phenotype to another exhibit the property of plasticity. It is increasingly clear that epithelial and endothelial cells enjoy some of this plasticity, which is easily demonstrated by studying the process of epithelial-mesenchymal transition (EMT). Published reports from the literature typically rely on ad hoc criteria for determining EMT events; consequently, there is some uncertainty as to whether the same process occurs under different experimental conditions. As we discuss in this Personal Perspective, we believe that context and various changes in plasticity biomarkers can help identify at least three types of EMT and that using a collection of criteria for EMT increases the likelihood that everyone is studying the same phenomenon -namely, the transition of epithelial and endothelial cells to a motile phenotype.

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Gastrulation events in the prestreak pig embryo: Ultrastructure and cell markers

Cited by 59 publications

References 53 publications

Development of a Noninvasive Monitoring System for Evaluation of Oct-3/4 Promoter Status in Miniature Pig Somatic Cell Nuclear Transfer Embryos

Development of a Noninvasive Monitoring System for Evaluation of Oct-3/4 Promoter Status in Miniature Pig Somatic Cell Nuclear Transfer Embryos

Pig Epiblast Stem Cells Depend on Activin/Nodal Signaling for Pluripotency and Self-Renewal

Biomarkers for epithelial-mesenchymal transitions

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