Intestinal cell proliferation is not altered in <i>agr2</i> morphants.

<p>Images of p-Histone H3-stained cells in the mid-intestines and posterior intestines of 104-hpf wild type embryos (A), agr2–5 mmMO1 and 5 mmMO2-coinjected embryos (B) and <i>agr2</i> morphants (C) are shown. Arrows indicate p-Histone H3-stained cells. (D) Comparison of the percentages of p-Histone H3-stained M phase cells among wild type embryos, agr2–5 mmMO1 and 5 mmMO2-coinjected embryos, and <i>agr2</i> morphants is shown. Error bars indicate the standard error. Scale bars represent 100 µm.</p

Enlarged areas of mature Alcian-blue stained goblet cells are detected in <i>agr2</i>-overexpressed embryos.

<p>Substantially increased areas of mature Alcian blue-stained intestinal goblet cells in 42% 104-hpf <i>agr2</i>-overexpressed (C, <i>n</i> = 29) embryos compared to <i>lacZ</i>-overexpressed (B, <i>n</i> = 20) embryos and wild type (A, <i>n</i> = 18) embryos were observed. Comparison of the area of Alcian blue-stained goblet cells in wild type, <i>agr2</i>- and <i>lacZ</i>-overexpressed embryos is shown (D). Arrows indicate examples of Alcian-blue stained goblet cells with enlarged areas. Student's <i>t</i>-test was conducted and *p<0.001. Scale bars represent 100 µm.</p

<i>agr2</i> morpholino antisense oligomer knockdown analyses.

<p>(A) Phenotype comparison among wild type, agr2–5 mmMO1 and 5 mmMO2-coinjected, and agr2-MO1 and agr2-MO2-coinjected embryos at 24 and 104 hpf. (B) Whole-mount immunohistochemistry demonstrates that coinjection of agr2-MO1 and agr2-MO2 prevents the synthesis of Agr2 protein in intestinal goblet cells. Confocal images of either wild type, agr2–5 mmMO1 and 5 mmMO2-coinjected, or agr2-MO1 and agr2-MO2-coinjected 104 hpf embryos were recorded under transmitted mode (a, d, g) or using 494/517 nm excitation/emission wavelengths (b, e, h). Merged images are shown (c, f, i). (C) Green fluorescence was not detected in agr2-MO1, agr2-MO2 and <i>CMV</i>-<i>agr2</i>-<i>mo</i>-<i>GFP</i> coinjected (c) 30 hpf embryos, whereas bright green fluorescence was observed in <i>CMV</i>-<i>agr2</i>-<i>mo</i>-<i>GFP</i>-injected (a) and agr2–5 mmMO1, agr2–5 mmMO2 and <i>CMV</i>-<i>agr2</i>-<i>mo</i>-<i>GFP</i> coinjected (b) 30 hpf embryos. Scale bars represent 100 µm.</p

Zebrafish <i>agr2</i> is solely expressed in intestinal goblet cells.

<p>Fluorescent whole-mount double <i>in situ</i> hybridization was conducted on 104-hours post fertilization (hpf) embryos using <i>agr2</i> (green) and <i>glucagon</i> (red) as RNA probes. Confocal images were recorded using excitation/emission wavelengths of 494/517 nm for fluorescein (A) and 550/570 nm for cyanine 3 (B). Merged image is shown (C). Arrows indicate <i>agr2</i>-expressing goblet cells and arrowheads specify <i>glucagon</i>-expressing enteroendocrine cells. Scale bars represent 100 µm.</p

Terminal differentiation of intestinal goblet cells is affected in <i>agr2</i> morphants.

<p>Significant increases in immature Alcian blue-stained goblet cell numbers were detected in 104- (C, <i>n</i> = 46) and 120-hpf (G, <i>n</i> = 23) <i>agr2</i> morphants compared to those in either 104- (A, <i>n</i> = 40) and 120-hpf (E, <i>n</i> = 30) wild type or in 104- (B, <i>n</i> = 41) and 120-hpf (F, <i>n</i> = 27) agr2–5 mmMO1 and 5 mmMO2-coinjected embryos. Inset shows a mature and an immature goblet cell. Comparison of both immature and mature goblet cell numbers among <i>agr2</i> morphants, wild type or agr2–5 mmMO1 and 5 mmMO2-coinjected embryos at 104 and 120 hpf are shown (D, H). A Student's <i>t</i>-test was conducted to compare immature goblet cell numbers in <i>agr2</i> morphants with those in wild type or agr2–5 mmMO1 and 5 mmMO2-coinjected embryos. *p<0.001. Scale bars represent 100 µm.</p

Transmission electron microscopy shows abnormal goblet cell structures in <i>agr2</i> morphants.

<p>Mid-intestinal images of 104- and 120-hpf wild type (A, D), agr2–5 mmMO1 and 5 mmMO2-coinjected (B, E), and <i>agr2</i> morphants (C, F) are shown. ER ultrastructure at higher magnification is shown in insets. Arrows indicate mature goblet cells and arrowheads denote immature goblet cells. Scale bars represent 2 µm.</p

Defective development of the nervous system in the hypo-Q/R-editing morphants.

<p>(<b>A</b>) The development of early and mature neurons. Confocal microscopic observations of the kaede fluorescence in Tg(<i>HuC:kaede</i>) receiving morpholino injections. The kaede-expressing domain is reduced in the brain of hypo-Q/R-editing morphants. CeP, cerebellar plate; cng, cranial ganglion; fb, forebrain; hb, hindbrain; pllg, posterior lateral line ganglion; r, retina; trg, trigeminal neuron. (<b>B</b>) The development of cranial motor neurons. Confocal microscopic observations of the GFP in the heads of Tg(<i>isl1:GFP</i>) receiving morpholino injections. The cranial motor neurons are only mildly affected. nIII; oculomotor nuclei; nV; trigeminal nuclei; nX, vagus nuclei. (<b>C</b>) The development of spinal motor neurons. Confocal microscopic observations of the GFP in the trunks of Tg(<i>isl1:GFP</i>) receiving morpholino injections. The spinal motor neurons dorsal to the yolk extension are shown. The effects of morpholino treatments were classified into three groups by relative density of motor neuron in each treatment. The numbers in parenthesis indicate the numbers of larvae in a class over all the observed larvae. Scale bar represents 100 µm. (<b>D</b>) The development of lateral line neuromasts. Epifluorescent microscopic observations of the lateral line neuromasts stained by Di-4-Asp <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097133#pone.0097133-Alexandre1" target="_blank">[48]</a>. L1-L5 are the posterior lateral line neuromasts, and tail represents the tail neuromasts. L1′ is the secondary PLL neuromast. The average ± s.d. (number of larva) of the primary lateral line neuromasts are shown below. Larvae without tail neuromast were excluded from the analysis.</p

Head morphology of hypo-Q/R-editing morphants.

<p>(<b>A)</b> Bright-field images of morphants established in the wild type background between 24 to 72 hpf. The genotypes of the morphants are indicated on the top, where <i>adar2MOc, adar2MOsp</i>, <i>adar2MOt</i>, <i>adar2MOt-p53^AUG</i> and <i>gria2αQRMO</i> respectively indicate embryos receiving the mismatch morpholino (MOc), splicing blocker (MOsp), translation blocker (MOt), MOt plus p53-MO^AUG<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097133#pone.0097133-Langheinrich1" target="_blank">[22]</a>, and RNA editing blocker (QRMO) morpholinos. Developmental time is shown on the left. The brain ventricles are enlarged and the size of heads is reduced in the hypo-Q/R-editing morphants. (<b>B</b>) Bright-field images of <i>p53</i> mutant (<i>tp53^zdf1</i>) receiving morpholinos described in (A) between 24 to 72 hpf. The morphological changes of <i>adar2MO</i> and <i>gria2αQRMO</i> cannot be suppressed by losing p53 activity.</p

Increased p53-dependent apoptosis in the hypo-Q/R-editing morphants.

<p>(<b>A</b>) Apoptosis at the 5-somite stage. Representative images, except those co-injected with p53-MO^AUG, were selected from the ones showing the mean fluorescence intensities in the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) analysis. The anterior is to the left, and dorsal side is to the top. Top panel shows the <i>adar2MOc</i>, <i>adar2MOsp</i> and <i>adar2MOt</i>. Middle panel shows <i>adar2MOt</i> co-injected with p53^AUG morpholino to block p53 activity, with <i>adar2</i> cRNA (Adar2) and with <i>adar2^cd</i> cRNA (Adar2^cd). The bottom panel shows <i>gria2αQRMO</i>, and the <i>gria2αQRMO</i> co-injected with p53^AUG and with <i>adar2</i> cRNA. (<b>B</b>) Tukey box plot of the relative TUNEL signals at 5-somite stage. Boxes represent 50% inter quartile values. Black and red lines respectively mark the median and mean intensities. Outliers are marked as dots. All signals were normalized to a wild-type embryo, showing mean intensity, stained in the same batch (relative fluorescence intensity). The total embryos included in the analysis are 8 <i>adar2MOc</i> (2 batches), 42 <i>adar2MOt</i> (9 batches), 19 <i>adar2MOt-p53^AUG</i> (3 batches), 20 <i>adar2MOt+</i>Adar2 (3 batches), 12 <i>adar2MOt+</i>Adar2<i>^cd</i> (3 batches), 9 <i>gria2αQRMO</i> (3 batches), 7 <i>gria2αQRMO-p53^AUG</i> (3 batches) and 5 <i>gria2αQRMO+</i> Adar2 (2 batches). (<b>C</b>) Increased p53-dependent apoptosis in specific regions of the 24- and 36-hpf hypo-Q/R-editing morphants. Lateral views of head and trunk at 24 and 36 hpf. Arrows indicate the apoptotic cells along the horizontal myoseptum. Scale bars represent 100 µm.</p

Gene expression in neural tube and migratory neural crest.

<p><b>(A, B)</b> Embryos are under lateral view. <b>(A)</b> Expressions of brain regionalization genes. Expression of <i>fgf8</i>, <i>krox20</i>, and <i>pax6a</i> appear normal in the 24 hpf hypo-Q/R-editing morphants. (<b>B, C</b>) Expressions of neural crest genes. The expression of mesenchymal condensations marker, <i>sox9a</i>, in the pharyngeal arch (pa, *) are slightly but consistently reduced in the hypo-Q/R-editing morphants. Expressions of neural crest markers <i>foxd3</i> and <i>crestin</i> are mildly affected at 6-somite and 16-somite stages. Anterior is respectively to the left and top at lateral and dorsal views. 1, 2 and 3 are the three migration cranial neural crest streams originated from posterior mesencephalon and hindbrain. Arrows indicate the midbrain hindbrain boundary. e, eye; hb, f, forebrain, hb, hindbrain; r2 and r4, rhombomeres 2 and 4.</p