<i>LIPH</i> expression at three different fetal stages (days 19, 25 and 29) corresponding respectively to the formation of the central primary, lateral primary and secondary hair follicles.

<p>(Bars = 20 µm). (A) Q-PCR expression of <i>LIPH</i> mRNA in the <i>orylag®</i> rabbit skin (light blue bars) and in the normal rabbit skin (dark blue bars). The Y axis represents the relative expression level of <i>LIPH</i>. (B) Histology (ROAN staining) of <i>orylag®</i> (B1) and normal rabbit skins (B2). (C) Immunohistochemistry of <i>orylag®</i> (C1) and normal rabbit skins (C2).</p

Primer pairs used for PCR amplification, qPCR and ISH.

<p>Primer pairs used for PCR amplification, qPCR and ISH.</p

<i>LIPH</i> expression in adult <i>orylag®+, orylag®−,</i> rex, heterozygous and normal rabbit skins.

<p>(Bars = 20 µm). (A) Q-PCR expression of <i>LIPH</i> mRNA. The Y axis represents the relative expression level of <i>LIPH</i>. (B) <i>In situ</i> hybridization. (C) Immunohistochemistry.</p

<i>LIPH</i> expression in <i>orylag®</i> and heterozygous skin of adult synchronized rabbits at the anagen and catagen/telogen follicle cycle stages.

<p>(Bars = 20 µm). (A) Q-PCR expression of <i>LIPH</i> mRNA. The Y axis represents the relative expression level of <i>LIPH</i>. (B) <i>In situ</i> hybridization. (C) Immunohistochemistry.</p

Lipase activity of wild-type <i>LIPH</i> and 1362delA <i>LIPH</i> mutant constructs.

<p>Values obtained are shown as a percentage of that observed in cells transfected with the empty vector (pcDNA3.1+, control).</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

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

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

Elongation classification and development success rates.

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