Development of the palatal shelves in E14.5 <i>Krt5</i>-tTA;pTRE-ΔNp63α bi-transgenic mice.

<p>(<b>A</b>) The horizontal palatal shelves of wild-type embryos have formed a midline epithelial seam which has started to degenerate. (<b>B</b>) In contrast, the palatal shelves of <i>Krt5</i>-tTA;pTRE-ΔNp63α embryos have approximated and adhered via a thickened epithelial midline. (<b>C,D</b>). BrdU immunostaining confirmed that the persistent MES continued to proliferate with no evidence of apoptosis (<b>E, F</b>). (<b>G-J</b>) Immunostaining revealed a persistent double layer of keratin 17-positive periderm cells and an underlying layer of p63-positive basal cells. Scale bars: 50 μm.</p

Loss of p63 results in adhesion defects in palatal epithelia.

<p>Immunofluorescence analysis of (<b>A</b>, <b>B</b>) plakoglobin, (<b>E</b>, <b>F</b>) nectin-1, and (<b>I</b>, <b>J</b>) nectin-4 reveals strong expression of these proteins at the junction between the periderm/basal cells in the palatal epithelia of E13.5 wild-type mice. (<b>C</b>, <b>D</b>, <b>G</b> and <b>H</b>) In contrast, plakoglobin and nectin-1 expression are markedly down-regulated in the E13.5 <i>p63</i>^-/- palatal epithelia. (<b>K</b> and <b>L</b>) Nectin-4 expression levels in <i>p63</i>^-/- palatal shelves are comparable to those of wild-type mice; however deconvolution images reveal that expression of nectin-4, which is normally restricted to the periderm/basal junction, is mis-localized in the epithelia of E13.5 <i>p63</i>^-/- palatal shelves and is expressed between adjacent basal cells. (<b>M</b>) qPCR analysis of palatal shelves dissected from E14.0 wild-type and <i>p63</i>^-/- mice indicates that <i>Pvrl1</i> transcripts are significantly reduced in <i>p63</i>^-/- palatal shelves while <i>Pvrl4</i> levels are comparable to wild-type.** = P <0.01, Mann Whitney U test, n = 5 for each genotype. Scale bars: A, C, E, G, I, K, 50 μm; B, D, F, H, J, L, 20 μm.</p

Down-regulation of p63 in the medial edge epithelia allows periderm migration.

<p><i>mKrt17</i>-GFP transgenic mice were used for time-lapse confocal imaging of periderm migration during development of the secondary palate. (<b>A-C</b>) On a wild-type background, GFP-positive periderm cells migrate out of the midline seam (arrowed) to form the epithelial triangles on the oral and nasal surfaces as part of the process whereby mesenchymal continuity across the palate is achieved. (<b>D-F</b>) In contrast, in <i>Tgfb3</i>^-/- embryos, GFP-positive periderm cells fail to migrate out of the midline epithelial seam and the secondary palate remains cleft. (<b>G-I</b>) Reducing p63 dosage in <i>Tgfb3</i>^-/- embryos restores the migratory periderm phenotype allowing palatal fusion and rescuing the cleft palate phenotype (region of midline is arrowed). p: palatal shelves. The images are representative stills taken at the same Z position over a 24-hour culture period. The videos are provided as Supplemental Material, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006828#pgen.1006828.s010" target="_blank">S1</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006828#pgen.1006828.s012" target="_blank">S3 Videos</a>.</p

p63 is essential for periderm development.

<p>(<b>A</b>) ChIP-qPCR validation of p63-bound sites within regulatory regions surrounding ‘cleft palate-associated genes’. Fold-enrichment for each binding region was calculated relative to a control region in exon 2 of myoglobin (set at 1; pale bar), to which p63 does not bind. (P) Genes associated with periderm formation; Intra, intragenic. Asterisks represent level of significance: * = P <0.05, ** = P <0.01, *** = P <0.001. Student’s t-test, n = 4. (<b>B</b>-<b>E</b>) Histological analysis of palatal epithelia at E12.5 and E13.5. (<b>B</b> and <b>D</b>) Coronal sections of wild-type palatal shelves at E12.5 and E13.5 reveal a layer of flattened periderm cells overlying the basal epithelia. (<b>C</b> and <b>E</b>) In <i>p63</i>^-/- palatal shelves, the flattened cells are absent and individual ‘bead-like’ cells are observed above the basal epithelia at E12.5 (<b>C</b>) and E13.5 (<b>E</b>). Boxed insets represent higher magnification of the respective regions. (<b>F</b>-<b>I</b>) Keratin 17 (K17) expression at E12.5 and E13.5. (<b>F</b>) K17 is expressed in a number of flattened cells overlying the basal epithelia in wild-type palatal shelves at E12.5. (<b>G</b>) In contrast, K17 is not detected in <i>p63</i>^-/- palatal epithelia at E12.5. (<b>H</b>) From E13.5, K17 expression is observed throughout the periderm layer in wild-type palatal epithelia. (<b>I</b>) However, K17 expression is absent in <i>p63</i>^-/- palatal shelves although ectopic K17 expression is detected in basal epithelial cells at E13.5 (arrow). Scale bars: 50 μm.</p

Rescue of cleft palate in <i>Tgfb3</i>^-/- mice by reducing p63 dosage in the medial edge epithelia.

<p>(<b>A</b>) The palatal shelves of wild-type mice elevate, adhere and fuse to form a transient midline epithelia seam at E14.5 while (<b>B</b>) the palatal shelves of <i>Tgfb3</i>^-/- embryos elevate but fail to adhere to, or fuse with, one another. (<b>C</b>) In contrast, reducing p63 dosage in a <i>Tgfb3</i>^-/- background restores the wild-type phenotype. (<b>D—I</b>) In E14.5 wild-type mice, p63 expression is down-regulated in the MEE cells which cease proliferation and undergo apoptosis. In contrast, in E14.5 <i>Tgfb3</i>^-/- mice, p63 expression is maintained in the MEE which continue to proliferate and do not undergo cell death. Reducing p63 dosage in a <i>Tgfb3</i>^-/- background restores wild-type behaviour in the MEE. (<b>J—L</b>) In wild-type and <i>Tgfb3</i>^-/-;<i>p63</i>^+/- mice, keratin 17-positive periderm cells migrate out of the MEE to form the oral and nasal epithelial triangles, whereas in <i>Tgfb3</i>^-/- mice, distinct keratin 14-positive basal and keratin 17-positive periderm layers persist and the palatal shelves fail to adhere/fuse. (<b>M—O</b>) <i>Mmp13</i>, which is absent from the MEE in E14.5 <i>Tgfb3</i>^-/- mice (arrowed), is restored to a wild-type expression pattern in <i>Tgfb3</i>^-/-;<i>p63</i>^+/- embryos. p: palatal shelves; t: tongue. Scale bars: A-C & M-O, 250 μm; D-L, 100 μm.</p

Molecular consequences of increasing p63 expression during palatal development.

<p>(<b>A, B</b>) The expression of Bcl11b (<b>A, B</b>), <i>Znf750</i> (<b>C, D</b>), <i>Jag2</i> (<b>E,F</b>) and <i>Perp</i> (<b>G, H</b>), all of which are down-regulated in the medial edge epithelia of wild-type mice, is maintained in the medial edge epithelia of their <i>Krt5</i>-tTA;pTRE-ΔNp63α bi-transgenic littermates. In contrast, <i>Mmp13</i> which is normally expressed in the midline epithelial seam of wild-type mice as they adhere until degeneration of the seam, is down-regulated in the medial edge epithelia of <i>Krt5</i>-tTA;pTRE-ΔNp63α embryos. p: palatal shelves; t: tongue. Scale bars: 200 μm.</p