Endogenous <i>Sox3</i> and the <i>Sox3</i> transgene are expressed in the developing and mature SCO.

<p><b>A–H:</b> Sagittal sections of 12.5 dpc (A–D) and 15.5 dpc (E–H) wild type (A,C,E,G) and <i>Nr</i>/+ (B,D,F,H) embryos immunostained for SOX3 (A,B,E,F) and EGFP (C,D,G,H). SOX3 is expressed in all cells of the wild type and <i>Nr/+</i> SCO. Note the presence of the EGFP signal and higher intensity of SOX3 signal in Nr/+ transgenic sections. <b>I–L:</b> Mid-sagittal sections of adult wild type (I,K) and <i>Nr</i>/+ (J,L) brains showing expression of SOX3 and EGFP. <b>M–P:</b> Coronal sections of 12.5 dpc wild type (M,O) and <i>Sr/+;Nr/+</i> transgenic (N,P) SCO tissue. In wild type embryos, a lower level of endogenous SOX3 was detected in the SCO primordium in comparison with periluminal cells flanking the SCO (asterisk in M). This difference in SOX3 signal intensity was not observed in <i>Sr/+;Nr/+</i> embryos (N). Arrows: PC (posterior commissure). Yellow arrowheads: points of invagination of the SCO primordium. White arrowhead: mesocoelic recess. In A–L, anterior is to the left and dorsal to the top. Scale bar: 200 µm.</p

<i>Sox3</i> transgenic mice displayed CH.

<p><b>A,B:</b> H&E stained coronal section of adult <i>Sox3</i> transgenic mouse brain without (A) and with (B) overt hydrocephalus. Note the expanded lateral ventricles (LV), thinning of cerebral cortex (double-headed arrow) and hippocampal deformation (arrows) in the CH founder. <b>C,D: </b><i>Nr/+</i> adult mice showing dome-shaped cranium (arrow) due to overt CH (D) and normal cranial morphology (C). <b>E,F:</b> Nissl stained 18.5 dpc coronal brain sections of wild type (E) and <i>Sr/+;Nr/+</i> (F) 18.5 dpc embryonic brains. Note the expansion of the lateral ventricles (LV) in the transgenic embryo (F). Scale bar: 2 mm (A–B) and 1 mm (E–F).</p

The role of ERK1/2, Src Family Kinases and p38 MAPK in the maintenance of EPL cells.

<p><b>A-D.</b> Photomicrographs of mES cells cultured in MEDII for 2 days and subsequently in MEDII containing medium supplemented with DMSO (A), 1 μM PD0325901 (B) 10 μM PP2 (C) and 10 μM SB203580 (D). Cells were stained for alkaline phosphatase activity (red/purple stain). Scale bar = 200 μm. <b>E, F.</b> mES cells were cultured in MEDII for 2 days and subsequently in MEDII containing medium supplemented with DMSO (■), DMSO with 1 μM PD0325901 (E, □), DMSO with 10 μM PP2 (F, □) or DMSO with 10 μM SB203580 (G, □). RNA from these cells was analyzed for expression of <i>Oct4</i>, <i>Sox2</i>, <i>Nanog</i>, <i>Rex1</i>, <i>Spp1</i>, <i>Gbx2</i>, <i>Dnmt3b</i> and <i>Otx2</i> by real-time PCR. Expression was normalized to <i>β-actin</i>. Error bars represent SEM; n = 3. EPL cells cultured in MEDII with inhibitor were compared to EPL cells in MEDII with DMSO, ** <i>p</i> ≤ 0.05, or mES cells, # <i>p</i>≤ 0.05.</p

The role of p38 MAPK in the formation of EPL cells in response to MEDII.

<p><b>A.</b> mES cells were pre-treated with 10 μM SB203580 for 60 minutes. 200 μM l-proline was added, as denoted and the cells incubated for a further 60 minutes. Cells were collected and analysed by western blot for the presence of phosphorylated pHspb2. Total Hspb2 was used as a loading control. <b>B, C.</b> mES cells were cultured medium supplemented with MEDII and DMSO (A) or MEDII and 10 μM SB203580 for 3 days. Scale bar = 200 μm. <b>D.</b> mES cells were cultured in medium supplemented with MEDII and DMSO (■) or MEDII and 10 μM SB203580 (□) for 3 days. RNA from these cells was analyzed for expression of <i>Oct4</i>, <i>Sox2</i>, <i>Nanog Rex1</i>, <i>Spp1</i>, <i>Gbx2</i>, <i>Dnmt3b</i>, <i>Otx2</i> and <i>Fgf5</i> by real-time PCR. Expression was normalized to <i>β-actin</i> and expressed relative to mES cells (<i>Fgf5</i> has been expressed relative to MEDII + DMSO). Error bars represent SEM; n = 4. mES cells + MEDII + SB203580 were compared to mES cells + MEDII + DMSO (** p ≤ 0.05) or mES cells (# p ≤ 0.05). <b>E.</b> mES cells were cultured in ES cell medium supplemented with MEDII, DMSO or MEDII and 10 μM 10 μM SB203580 for 3 days and formed into EBs. EBs were collected on days 2, 3 and 4, RNA was isolated and analyzed for expression of <i>T</i> and <i>Gapdh</i> by RT-PCR. n = 3.</p

Inhibition of MEK1 prevents the formation of EPL cells in response to MEDII.

<p>A. mES cells were pre-treated with 1 μM PD0325901 for 60 minutes. 200 μM l-proline was added, as denoted, and the cells incubated for a further 60 minutes. Cells were collected and analysed by western blot for the presence of phosphorylated ERK1 or ERK2. Total ERK1/2 was used as a loading control. The intensity of the pERK2 band was measured using Quantity One software (BioRad) and represented as a proportion of total ERK1/2. Error bars represent SEM; n = 4; * <i>p</i> ≤ 0.05 when compared to mES cells. B-C. mES cells were cultured in MEDII- and DMSO-containing medium (B) and MEDII- and 1μM PD0325901-contianing medium (C) for 3 days. Scale bar = 200 μm. D. MEDII- and DMSO-containing medium (■) and MEDII- and 1μM PD0325901-contianing medium (□) for 3 days. RNA from these cells was analyzed for expression of <i>Oct4</i>, <i>Sox2</i>, <i>Nanog</i>, <i>Rex1</i>, <i>Spp1</i>, <i>Gbx2</i>, <i>Dnmt3b</i>, <i>Otx2</i> and <i>Fgf5</i> by real-time PCR. Expression was normalized to <i>β-actin</i> and expressed relative to mES cells (<i>Fgf5</i> has been expressed relative to MEDII + DMSO). Error bars represent SEM; n = 3. mES cells + MEDII + PD0325901 were compared to mES cells + MEDII + DMSO (** p ≤ 0.05) or mES cells (# p ≤ 0.05).</p

Structure, copy number and expression of the <i>Sox3</i> transgene.

<p><b>A:</b> Schematic representation of the transgene showing the <i>Sox3</i> coding sequence (which is contained in a single exon) and the <i>IRES</i>-<i>EGFP</i> reporter cassette and flanking genomic sequence (not to scale). IRES indicates the internal ribosome entry site. <b>B:</b> qPCR analysis of transgene copy number in the <i>Sr/+</i> and <i>Nr/+</i> lines demonstrating that they contain 2 and 1 copy of the transgene, respectively. <i>Sox3</i> null (KO) genomic DNA was used as a negative control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029041#pone.0029041-Rizzoti1" target="_blank">[22]</a>. <i>Sox1</i> quantification was performed as an endogenous autosomal control. <b>C:</b> Quantitative fluorescence Western blot analysis showing a 2–3-fold increase in SOX3 protein levels in 10.5 dpc <i>Sr/+</i> and <i>Nr/+</i> embryos.</p

Dose-dependent penetrance of overt CH in <i>Sox3</i> transgenic mice.

<p>• and ∧: Significantly fewer progeny were present at weaning (<i>*p</i> = 0.030, ∧<i>p</i> = 0.001; Chi Square test).</p><p>Table showing the penetrance of overt CH in <i>Sr/</i>, <i>Nr/+</i> and <i>Sr/+;Nr/+</i> pups at weaning (approximately 3 weeks).</p

Defective SCO development in <i>Sr/+;Nr/+</i> embryos from 13.5 dpc.

<p><b>A–D:</b> Nissl stained sagittal sections of 12.5 dpc wild type (A,C) and <i>Sr/+;Nr/+</i> (B,D) SCO primordia showing similar morphology and cell body density. C and D are boxed regions in A and B, respectively. <b>E–H:</b> Nissl-stained sagittal sections of 13.5 dpc wild type (E,G) and <i>Sr/+;Nr/+</i> (F,H) SCO region. Note the thinner SCO primordium and lack of pineal recess in the <i>Sr/+;Nr/+</i> embryo. <b>I–N:</b> Nissl-stained sagittal (I,J) and coronal (K–N) sections of 15.5 dpc wild type (I,K,M) and <i>Sr/+;Nr/+</i> (J,L,N) SCO region. Note the severely disrupted PC and absence of pseudostratified ependymal layer of the SCO in the <i>Sr/+;Nr/+</i> embryo. M and N are boxed regions in K and L, respectively. <b>O–P:</b> RF (red) and SOX3 (green) immunostaining of 15.5 dpc sagittal wild type (O) and <i>Sr/+;Nr/+</i> (P) sections. <i>Sr/+;Nr/+</i> transgenic embryos maintain SOX3 expression in the dorsal midline but fail to generate RF. Arrows point to the SCO invagination in the roof plate and the double-headed arrow to the mesocoelic recess. The hash sign indicates the SCO remnant. The asterisk indicates the approximate position where the pineal gland should develop in <i>Sr/+;Nr/+</i> embryos. The arrowhead indicates the pineal recess. EpTh: epithalamus. MHN: medial habenular nucleus. Pt: pretectum. FR: fasciculus retroflexus. PC: posterior commissure. In A–H and K–P, anterior is to the left and dorsal to the top. Scale bars are 100 µm (C–F, M,N), 200 µm (A,B,G,H,K,L) and 1 mm (I,J).</p

The regulation of progression of the pluripotent lineage in culture.

<p>The cell states represented in vitro, naïve mES cells, primed mES cells and EPL cells have been aligned with the expression of Nanog and Otx2 and with their deduced intracellular signaling activity. Inducers of lineage progression are shown in orange; Calcineurin exerts its effects through dephosphorylation of NFAT and promotes NFAT translocation to the nucleus.</p

Summary of inhibitors used in this study.

<p>Summary of inhibitors used in this study.</p