The parapineal and habenular defects are coupled in <i>sox2</i> morphants.

<p>(<b>A–C</b>) In control embryos, the left-sided parapineal projects towards the left habenula. As a result, the left habenula has denser neuropils than the right, as judged by phalloidin staining. (<b>D–F</b>) In <i>sox2</i> morphants with left-sided parapineal projections, the left habenula is larger than the right. (<b>G–I</b>) Morphants with right-sided parapineal organs display reverse habenular asymmetries, whereas (<b>J–L</b>) morphants with bilateral parapineal projections have symmetric habenulae. (<b>M</b>) The average volume of the left (blue bars) and right (red bars) habenular neuropils, as judged by the volume of phalloidin-positive areas within the habenulae. y-axis show volume in µm³. (<b>N</b>) Average asymmetry index in control (purple bar) and <i>sox2</i> morphants (orange bars). 3D reconstructions of confocal images at 4 dpf, arrows show parapineal projections and blue lines surround the habenular neuropils, error bars represent ± standard error, (<b>M</b>) * = p-value <0.05 and ** = p-value <0.001 (Wilcoxon test).</p

Sox2 expression within the pineal anlage is downregulated with differentiation.

<p>(<b>A–D</b>) Sox2 expression overlaps with expression of <i>flh</i>, a marker for pineal precursors, at 8 ss. (<b>E–H</b>) As pineal cells start differentiating, Isl1 is upregulated whereas Sox2 is downregulated. Yellow arrows show cells that have both Isl1 and Sox2 expression at low levels. (<b>I–L</b>) Sox2 is absent from the fully differentiated pineal cells. Scale bars = 25 µm, optical sections from confocal microscopy, insets show a three times magnified view of the image, (<b>E,I,M</b>) GFP expression of <i>Tg(flh:GFP)</i> (<b>F,J,N</b>) immunofluorescence for Isl1, (<b>G,K,O</b>) immunofluorescence for Sox2, (<b>H,L,P</b>) merged images with <i>Tg(GFP:flh)</i> in green, Isl1 in blue and Sox2 in red, developmental stages are shown at the beginning of each row.</p

Sox2 inhibits the PhR cell fate.

<p>(<b>A–C</b>) <i>Tg(aanat2:GFP)</i> drives GFP expression in the pineal PhRs. (<b>B</b>) Isl1 labels the pineal cells. (<b>D–F</b>) Knockdown of <i>sox2</i> results in increased number of PhRs. (<b>G</b>) The average number of PhRs in control (purple bar) and <i>sox2</i> morphants (orange bar). (<b>H–J</b>) <i>Tg(elavl3:GFP)</i> drives GFP expression specifically in the PNs. (<b>K–M</b>) The knockdown of <i>sox2</i> does not affect the number of PNs. (<b>N</b>) The average number of PNs in controls (purple bar) and <i>sox2</i> morphants (orange bar) does not significantly differ. Confocal maximum projections of 28 hpf embryos, scale bars = 25 µm, error bars represent ± standard error, ** = p-value <0.001 (MWU test). See also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s004" target="_blank">Figure S4</a></b>.</p

Knockdown of <i>sox2</i> results in upregulation of neurogenesis.

<p>(<b>A–B</b>) Neurogenesis is increased in <i>sox2</i> morphants, as judged by Isl1 expression. (<b>C</b>) Average number of Isl1-positive cells in control (purple bar) and <i>sox2</i> morphants (orange bar), at 24 and 28 hpf. (<b>D–E</b>) At 3–4 ss, two <i>flh</i>-domains are observed on either side of the neural plate. (<b>F–G</b>) By 7–8 ss, the two domains fuse to form the presumptive pineal gland. (<b>H–K</b>) In <i>sox2</i> morphants, <i>flh</i> is expressed in broader domains in relation to control siblings. Scale bars = 25 µm, (<b>A–B</b>) confocal maximum projections, (<b>C</b>) error bars represent ± standard error, ** = p-value <0.001 (MWU test). (<b>J,L,N,P</b>) dorsal views with anterior to the top, (<b>K,M,O,Q</b>) frontal views. See also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s002" target="_blank">Figure S2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s003" target="_blank">S3</a></b>.</p

A model for pineal cell-fate determination.

<p>Cell-fate determination has two phases: a prepatterning phase, followed by a determination phase. Sox2 (Sox2 levels or Sox2 along with a partner protein (X)) is important during the prepatterning phase, where is inhibits cells from adopting a PhR fate. In contrast, the Notch and BMP pathways are important during the determination fate, where BMP induces the PhR fate and Notch inhibits the PN fate. A yet-to-be-identified modulator (Y) is responsible for inducing the PN fate.</p

Knockdown of <i>sox2</i> results in abnormal parapineal development and disruption of the habenular asymmetries.

<p>(<b>A</b>) <i>gfi1ab</i> is expressed in parapineal cells on the left side of the brain in control embryos. (<b>B–D</b>) <i>sox2</i> morphants are categorized into three groups according to <i>gfi1ab</i> expression: left-sided expression (<b>B</b>), right-sided (<b>C</b>) and embryos with scattered <i>gfi1ab</i> cells (<b>D</b>). (<b>E</b>) <i>kctd12.1</i> is asymmetrically expressed in the habenulae, with a broader expression domain in the left than the right habenula. (<b>F–H</b>) In <i>sox2</i> morphants <i>kctd12.1</i> expression can be: asymmetric with more on the left side similar to control embryos (<b>F</b>), asymmetric with more on the right side (<b>G</b>) or symmetric (<b>H</b>). (<b>I</b>) A table showing the percentage of embryos with normal, reversed or bilateral parapineal organs, using different staining methods. Scale bars = 25 µm. See also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s009" target="_blank">Figure S9</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s010" target="_blank">S10</a></b> and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s015" target="_blank">Movie S4</a>.</b></p

Sox2 and Notch have synergistic effect on neurogenesis and complementary effects on cell-fate determination.

<p>(<b>A–D</b>) The number of pineal neurons (Isl1-positive cells) is increased in <i>sox2</i> morphants (<b>B</b>) and in DAPT-treated embryos (<b>C</b>), when compared to DMSO-treated controls (<b>A</b>). A synergistic effect is observed when both <i>sox2</i> and Notch are knocked down (<b>D</b>). (<b>E–H</b>) <i>Tg(aanat2:GFP)</i> drives GFP expression in the pineal PhRs in DMSO-treated controls (<b>E</b>). The number of PhRs is increased in <i>sox2</i> morphants (<b>F</b>), but remains unaffected in DAPT-treated embryos (<b>G</b>). The knockdown of both <i>sox2</i> and Notch results in an increased number of GFP-positive cells (<b>H</b>), comparable to the one observed in <i>sox2</i> morphants. (<b>I–L</b>) <i>Tg(elavl3:GFP)</i> expresses GFP in PNs. There is no difference in the number of GFP-positive in <i>sox2</i> morphants (<b>I</b>) when compared to DMSO-treated controls (<b>I</b>). The knockdown of Notch alone (<b>K</b>) or simultaneously with <i>sox2</i> (<b>L</b>) results in similar increase in GFP expression. (<b>M–O</b>) Average number of Isl1-positive cells (<b>M</b>), PhRs (<b>N</b>) and PNs (<b>O</b>) in untreated controls, DMSO-treated controls, untreated <i>sox2</i> morphants, DMSO-treated <i>sox2</i> morphants, DAPT-treated embryos and DAPT-treated <i>sox2</i> morphants. Confocal maximum projections of 28 hpf embryos, scale bars = 25 µm, error bars represent ± standard error, * = p-value <0.05 (MWU test), ** = p-value <0.001 (MWU test). See also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s005" target="_blank">Figure S5</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s007" target="_blank">S7</a></b> and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s012" target="_blank">Movie S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s013" target="_blank">S2</a></b>.</p

Suboptimal doses of <i>sox2</i> morpholinos result in increased number of PhRs and abnormal positioning of the parapineal organ.

a<p>Average number of PhRs (± standard error, number of embryos analyzed).</p>b<p>Percentage of embryos with left, right or bilateral parapineal projections (number of embryos in each category).</p

Sox2 controls PhR cell fate independently of BMP.

<p>(<b>A</b>) The transgenic line <i>Tg(aanat2:GFP)</i> marks the PhRs. (<b>B</b>) The transgenic line <i>Tg(BRE:GFP)</i> is as a BMP signaling reporter. (<b>C</b>) In <i>sox2</i> morphant embryos, the number of PhRs is increased at 24 hpf, when compared to controls (<b>A</b>). (<b>D</b>) The number of BMP-responsive cells at 24 hpf is unaffected when <i>sox2</i> is knocked down. (<b>E–F</b>) The number of PhRs (<b>E</b>) and BMP-responsive cells (<b>F</b>) is similar in control embryos at 28 hpf. (<b>G–H</b>) The number of PhRs (<b>G</b>) is higher than the number of BMP-responsive cells (<b>H</b>) at 28 hpf, in <i>sox2</i> morphants. (<b>I</b>) The average number of PhRs (<i>Tg(aanat2:GFP)</i>-positive cells) and BMP-responsive cells (<i>Tg(BRE:GFP)</i>-positive cells) in control and <i>sox2</i> morphant embryos, at 24 and 28 hpf. Confocal maximum projections, scale bars = 25 µm, error bars represent ± standard error, ** = p-value <0.001 (MWU test). See also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s008" target="_blank">Figure S8</a></b> and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s014" target="_blank">Movie S3</a></b>.</p