Pluripotency in the WT and <i>Prnp</i>-null cell lines.

<p>WT EB mRNA (<b>A</b>) and KO EB mRNA (<b>B</b>) expression patterns of three pluripotency genes during differentiation. Note the lack in the KO line (<b>B</b>) of an increase in <i>Nanog</i> expression on Day 5. C) WT EB mRNA expression pattern of <i>Prnp</i> during differentiation (Days 0, 1, 2, 3, 5, 7 and 13). <i>Prnp</i> is expressed at its highest levels on Day 5 (Error bars, s.e.m.).</p

Differentiation transcription behaviour of the WT and <i>Prnp</i>-null cell lines.

<p>mRNA expression patterns of PRNP related proteins (Doppel and Shadoo) and early markers of endoderm (<i>Hnf3</i>), mesoderm (<i>Brachyury -T-</i>) and ectoderm (<i>Nestin</i>) in WT and KO stem cells and in WT, <i>WT-like</i> KO and <i>PGC-like</i> KO EBs during differentiation (Days 5, 7 and 13). (* indicates statistical differences for the transcription of each gene between WT and KO ESC at P<0.05) (Error bars, s.e.m).</p

Primers used for RT-PCR.

<p>Primers used for RT-PCR.</p

<i>Prnp</i> and <i>Nanog</i> expression in testicles, ovaries and brain in adult, neonates and foetal mice at embryonic day 16.5 (E16.5).

<p>Note that correlative <i>Prnp-Nanog</i> expression only occurs in gonadal tissues. (Error bars, s.e.m.).</p

Consequences of <i>Prnp</i> absence in the BMP pathway.

<p>A) Detection of PGC markers (<i>Bmp4, Fragillis, Stella, Mvh4 and Dazl</i>) in WT ESC, WT GS and the two different <i>Prnp</i> KO ESC lines (KO 1 and KO 2) by PCR. Knock out cells show all the PGC markers analyzed in stem cell state. B) Diagram representing the BMP pathway showing the early, intermediate and later PGC markers (figure adapted from Young et al. 2009). C) Detection of primordial germ cell markers (<i>Bmp4, Fragillis, Stella, Mvh4 and Dazl</i>) by PCR during differentiation in WT EBs, WT-<i>like</i> KO EBs and PCG-<i>like</i> KO EBs.</p

Blocking of PRNP by the monoclonal antibody Sha31.

<p>On Day 5 and after 6 hours of antibody treatment, it is revealed that <i>Nanog</i> mRNA transcription is PRNP-dependent during differentiation and independent of feedback from the pluripotency cluster genes <i>Oct3/4</i> and <i>FoxD3</i>. (Error bars, s.e.m.).</p

Additional file 1: of Differential isoform expression and alternative splicing in sex determination in mice

(A) Summary of RNA sample, RNA-seq data, and number of genes and transcripts detected. (B) Summary of differentially expressed genes (DEGs) detected (P < 0.01). (C) Summary of differentially expressed isoforms (DEIs) detected (P < 0.01). (D) Comparison between DEGs and DEIs detected. (E) Summary of alternative splicing events detected. (DOCX 26 kb

LIF pathway regulation in the WT and <i>Prnp</i>-null cell lines.

<p>WT EB mRNA (<b>A</b>) and <i>PGC-like</i> KO EB mRNA (<b>B</b>) expression patterns of some LIF pathway genes during differentiation (Days 0 [(Stem Cell], 5, 7 and 13). (*indicates significant differences for a particular gene between WT and <i>PGC-like</i> KO EBs (P<0.05)) (Error bars, s.e.m.).</p

Apoptotic and macroscopic effects produced by the delection of <i>Prnp</i>.

<p>A) Comparing the numbers of EBs in the WT and KO lines (* P<0.05. Error bars, s.e.m.; “Y” axis represents “number of EBs per plate”) B) TUNEL analysis of wild type (WT) and <i>Prnp</i>-null (KO) embryoid bodies (EBs). C) Morphology of EBs on Day 7 and Day 80 of differentiation. The KO ESC line produced 2 types of EBs: one similar to WT (*) and the other called PGC-<i>like</i> KO EBs (arrowheads).</p

Metabolic transcription behaviour of the WT and <i>Prnp</i>-null cell lines.

<p>WT, <i>WT-like</i> KO EB and <i>PGC-like</i> KO EB mRNA expression patterns for <i>Irs2</i> (Insulin Receptor Substrate 2), <i>Slc2a</i> (Glucose Transporter 1 - <i>Glut1</i> -), <i>Gapdh</i> (Glyceraldehyde 3-phosphate Dehydrogenase) and <i>Sod1</i> (Superoxide Dismutase 1) on Days 5 (A) and 7 (B). (Error bars, s.e.m.).</p