Elongation classification and development success rates.

<p>Elongation classification and development success rates.</p

New biological outcomes of validated DEGs.

<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

Differential clustering of differentiating tissues and somatic cells.

<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

<i>KLF4</i> and <i>ACVR2A</i> discriminate among normal and abnormal SCNTs.

<p>A) Box plots of <i>KLF4</i> and <i>ACVR2A</i> expression in normal and abnormal SCNTs. B) PCR results from normal and abnormal EE tissues, pooled as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038309#pone.0038309.s002" target="_blank">Table S1</a>. C) Expression of <i>KLF4</i> and <i>ACVR2A</i> in the fibroblasts (5538, 7711 and 0029) used to generate SCNT High, Med, and Low conceptuses. Triangles correspond to cell passages temporally proximate to biopsy (p2-p3), squares to the passages closer to nuclear transfer (p7–p8).</p

Molecular uncoupling of E and EE tissues in SCNT conceptuses.

<p>The gene set used to classify each conceptus includes six genes from the extra-embryonic tissues and has been described as an accurate predictor of embryonic stages in controls. A) Patterns resulting from AI: examples of a filamentous conceptus with a disc at stage 3 (Fil-N2) and a trophoblastic vesicle (TV) with an ablated disc since Day 15. B) Patterns resulting from SCNT: examples of tubular conceptuses with a delayed embryonic disc (stage 2) or no disc (Ab2).</p

Uncoupling during elongation and gastrulation.

<p>Mild uncoupling events are in italics, severe uncoupling events are in bold, and coordinated E/EE differentiations are in normal font. Coordinated E/EE differentiations include normal/normal, delayed/delayed, and abnormal/abnormal morphologies.</p

Gastrulation patterns.

<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.

<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.

<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