28 research outputs found

    Uncoupled Embryonic and Extra-Embryonic Tissues Compromise Blastocyst Development after Somatic Cell Nuclear Transfer

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    Somatic cell nuclear transfer (SCNT) is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each); one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular “uncoupling”. Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538), we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity) and subsequent pregnancy loss. Finally, because it alters re-differentiation processes in vivo, SCNT reprogramming highlights temporally and spatially restricted interactions among cells and tissues in a unique way

    Knockdown of Brm

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    The SWI/SNF (SWItch/Sucrose NonFermentable or BAF, Brg/Brahma-associated factors) complexes are epigenetic modifiers of chromatin structure and undergo progressive changes in subunit composition during cellular differentiation. For example, in embryonic stem cells, esBAF contains Brg1 and Baf155, while their homologs, Brm and Baf170, are present in BAF of somatic cells. In this study, we sought to determine whether Brm and Baf170 play any roles in induced pluripotent stem cell (iPSC) reprogramming by using shRNA-mediated knockdown studies in the mouse model. We found that knocking down Brm during early, mid, and late stages (days 3, 6, and 9 after initial iPSC induction) and knocking down Baf170 during late-stage (day 9) reprogramming improve the numbers of iPSC colonies formed. We further showed that inhibition of these somatic BAF components also promotes complete reprogramming of partially reprogrammed somatic cells (pre-iPSCs). Finally, we found that the expression of Brm and Baf170 during reprogramming was regulated by Jak/Stat3 activity. Taken together, these data suggest that inhibiting somatic BAF improves complete reprogramming by facilitating the activation of the pluripotency circuitry

    Analyzing bovine OCT4 and NANOG enhancer activity in pluripotent stem cells using fluorescent protein reporters.

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    Green fluorescent protein (GFP) reporters controlled by the regulatory region of OCT4 and NANOG-two master regulators for pluripotency are widely used in studies of pluripotent stem cell establishment and embryo development. Alongside the challenge in establishing bovine pluripotent stem cells, the application of bovine-specific gene reporters has rarely been explored. Using lentivirus-based GFP reporter, we investigated the upstream regulatory regions of bovine OCT4 and NANOG. These reporters show activity in both naïve- and primed-state pluripotency when infected into mouse and human embryonic stem cells (ESCs), respectively. Consistent with what is found in humans and mice, the bovine OCT4-distal enhancer (bOCT4-DE) but not the proximal enhancer (bOCT4-PE) region is preferentially activated in naïve-state pluripotency. Furthermore, the bOCT4-DE region is silenced upon conversion of naive-state ESCs into primed-state epiblast stem cells (EpiSCs). Co-infection of mouse fibroblasts with the reprograming factors for induced pluripotent stem cell (iPSC) induction leads to the generation of GFP positive colonies, demonstrating that these GFP reporters can serve as live indicators for induced pluripotent cell establishment. We further proved that the bovine OCT4 distal enhancer is active in bovine blastocysts. We established the lentiviral-based fluorescent reporters controlled by bovine OCT4 and NANOG enhancer sequences. These reporter constructs show activity in naïve- and primed-pluripotent states. These reporters may serve as versatile tools for bovine ESC/iPSC generation and identification, as well as for developmental studies of bovine embryos

    Differential clustering of differentiating tissues and somatic cells.

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    <p>Ranking of SCNTs and controls based on the EE profiles (A) or the E stages (B). Ranking of the fibroblasts (5538, 7711 and 0029) based on their molecular profiles and using the 500 most variant genes among them. The cell passages (p) more temporally proximate to biopsy (p2 to p3) versus nuclear transfer (p7 to 8) were compared. Each ranking series is represented by a dendrogram.</p

    New biological outcomes of validated DEGs.

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    <p>A) <i>FN1</i> is restricted to the endoderm. <i>In situ</i> hybridisation in AI (a), IVP (b), and SCNT D18 EE tissues: Med (c), Low (d), High (e), using an anti-sense FN1 DIG-labelled riboprobe. (t) trophoblast, (e) endoderm. In the AI panel, f) shows c93/<i>SOLD1</i>, a trophoblast-specific control from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038309#pone.0038309-Degrelle1" target="_blank">[23]</a>. The sense probe gave a negative signal in all tissues (not shown). Despite differential expression levels in array and QPCR data, this gene is expressed in the same cells, regardless of conceptus origin. Only a small part of each conceptus is shown. Scale bar is 150 µm. B) Microvilli abnormalities in SCNT EE tissues at D18. In the SCNT groups where <i>MYO6</i> and <i>LHFPL2</i> were underexpressed (b, c), epithelial microvilli appeared shorter and/or fused. SEM images of the external face (extra-embryonic ectoderm or trophoblast) of D18 EE tissues from SCNT Low and Med conceptuses as compared to controls (AI in a). Magnifications: a) x 30000, b) x 35000, c) x 30000. C) <i>PLIN2</i> is expressed in the trophoblast of D18 EE tissues and absent from the yolk sac at D25 [the yolk sac is composed of endoderm (e) and mesoderm (m)]. It is also expressed in binucleated cells (BNC) from D63 bovine placentas. BNC are differentiated trophoblast cells, often considered as the anatomical equivalent of mouse giant cells. <i>In situ</i> hybridisations with an anti-sense PLIN2 DIG-labelled riboprobe on tissue cross-sections from D18 EET (a), D25 Yolk sac (b) and D63 placentas (c) developed after AI. The sense probe gave a negative signal in all tissues; data are not shown. Only a small part of each tissue is shown. Scale bar is 100 µm.</p

    Gastrulation patterns.

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    <p>A) Definition of gastrulation classes. Normal <i>Brachyury</i> patterns are shown in N1 (a) and D (b) embryonic discs. Abnormal <i>Brachyury</i> patterns (U-shaped and broadened labelling) are shown in Ab1 (c) embryonic discs. These are whole-mount <i>in situ</i> hybridisations with an anti-sense Brachyury DIG-labelled riboprobe performed on embryonic discs from two SCNT High (a, b) and two SCNT Low conceptuses (c, right and left panels). Scale bar: 100 µm. B) Overview of all conceptuses.</p

    Differentially expressed genes (DEGs) among Day 18 EE tissues.

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    <p>A) Paired comparisons according to SMVar (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038309#pone.0038309.s002" target="_blank">Table S1</a>). Across all the comparisons performed, 95 statistical occurrences were identified that corresponded to 72 unique DEGs. Multiple occurrences are in bold. Each gene ID is provided as a HUGO term. Among these DEGs, a few had been previously reported: single genes (<i>TUB1A1</i>, <i>B4GALT1)</i> or genes from the <i>CCDC, HSP or TKDP</i> families <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038309#pone.0038309-Kato1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038309#pone.0038309-RodriguezAlvarez1" target="_blank">[19]</a>. B) IPA networks. The DEG list was analyzed with the Ingenuity Pathway Analysis software to identify the top gene networks and the pathways connecting them. SCNT-specific differences (in red) were found in three of four networks. In the IPA database, 4 proteins were located in the extra-cellular space, 8 at the plasma membrane, 20 in the cytoplasm, and 24 in the nucleus, of which 8 were recognised as transcription regulators.</p

    Validated gene expression differences.

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    <p>The gene expression differences are presented according to their decreasing rank (or adjusted P-value) within the SMVar output lists. In A: differences between SCNT and AI, in B: differences between SCNT and IVP, in C: differences among SCNT. The y-axis of the each histogram corresponds to the relative expression values of each DEG in EE tissues (AI, IVP and SCNT High, Med, Low). Array data are in grey, QPCR data in black.</p
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