Malformation of the cranial cartilages of hypo-Q/R-editing morphants.

<p>Representative images of Alcian blue staining of the head cartilages are presented in three views. Ventral view is taken at a deeper focus from the dorsal side. Abbreviations: bh, basihyal; cb, ceratobranchials; ch, ceratohyal; ep, ethmoid plate; hs, hyosymplectic; m, Meckel's cartilage; not, notochord; pch, parachordal; pq, palatoquadrate; tr, trabeculae.</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

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

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

Injection of MOsp affects the splicing of <i>adar2</i> transcript.

<p>(<b>A</b>) 5′ gene structure of <i>adar2</i>. The gene structure is assigned by Slavov and Gardiner <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097133#pone.0097133-Slavov2" target="_blank">[14]</a>. The non-translated and translated exons are respectively depicted in open and filled rectangular boxes. The gray box depicts the 47-bp alternatively spliced exon. Editing of the A upstream to the gray box results in the alternative splice to include the 47 bp sequence (+47 transcript). The locations of annealing sites of antisense morpholinos, MOsp and MOt, and PCR primers to check the effects of MOsp are indicated as lines and arrows below the gene structure, respectively. (<b>B</b>) Splicing of <i>adar2</i> is perturbed by injecting MOsp. RT-PCR was performed with RNA extracted from wild type (W) and <i>adar2MOsp</i> (M) of various development stages (hpf) shown on top of ethidium-bromide-stained agarose gel. An equal amount of cDNA that gives rise to the same amount of <i>actb1</i> amplicon was used to amplify <i>adar2</i>. Sizes of molecular markers are shown on the left side of the gel. Asterisk and double-asterisk respectively indicate the products of normal and edited (+47) <i>adar2</i>. Arrow indicates major aberrant splice products.</p

Quantitative analysis of gene expression^a at 24 hpf.

a<p>: a relative gene expression level was determined and then normalized to the expression level of β-actin (actb1). The values (mean ± standard deviation) are the relative gene expression levels of morphants compared to those of the wild type (un-injected) of the same batch.</p><p>* indicates significant difference (p<0.05) between hypo-Q/R-editing morphants and adar2 control morphant (adar2MOc)</p><p>** indicates significant difference between adar2MOt-p53^AUG and adar2MOt.</p>b<p>: Accession numbers are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097133#pone.0097133.s003" target="_blank">Table S1</a>. neuroD and neurog1 (neurogenin 1) are proneuron genes; ccng1(cyclin G1), mdm2 and Δ113p53 (a short isoform of p53 transcribed from an internal promoter) are p53-responsible genes. 3′-p53 and 5′-p53 respectively represent transcripts encoding all the isoforms of p53 and long isoform of p53. sox9a and sox9b are expressed in the neural crest cells and other cell types.</p>c<p>: n, number of independent injection</p

Expression patterns of zebrafish <i>adar2</i> during embryogenesis.

<p>The developmental stages are indicated on top and on the left, as hour post fertilization (hpf). (<b>A, B, C, G, I, K, M and O</b>) Lateral views and (<b>D, E, F, H, J, L, N, P and P’</b>) dorsal views of the embryos. The anterior and dorsal sides are respectively to the left and top. (<b>P and P’</b>) Images were taken from two different focuses. P’ Image is slightly deeper showing the <i>adar2</i> expression in the ventral structures. Abbreviations: cb1-5, ceratobranchials 1–5; CeP, cerebellar plate; cng, cranial ganglion; di, diencephalon; fb, fin bud; gc, retinal ganglion cells; h, heart; hb, hindbrain; Hy, hypothalamus; Inl, inner nuclear layer; L, liver; mo, medullar oblongata; pf, pectoral fin; pllg, posterior lateral line placode/ganglion; r, retina; sc, spinal cord; t, telencephalon; T, tegmentum; TeO, tectum opticm; Th, thalamus.</p

Efficiencies of Q/R RNA editing of gria2α.

a<p>: morphants and protein expressed from the injected cRNA.</p>b<p>: The fractions of edited <i>gria2α^R</i> in the total <i>gria2α</i> transcript were determined and then normalized to that of the un-injected (wild type) embryos of the same batch. The fractions of <i>gria2α^R</i> present in the 12-hpf wild type embryos varied from 50% to 65%, while it was between 96 to 98% in the 24-hpf and 48-hpf wild type embryos <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097133#pone.0097133-Hoppmann1" target="_blank">[18]</a>. Values are represented as means ± standard deviation (number of independent injection). Statistics was analyzed by Student's <i>t</i>-test. Asterisk (*) indicates a significant difference (<i>p</i><0.05) versus <i>adar2MOc</i>. Double asterisks indicate a significant difference versus <i>adar2MOt</i>.</p>C<p>: the <i>gria2α^R</i> frequency of the corresponding <i>adar2MOsp</i> was 81%.</p>d<p>: the <i>gria2α^R</i> frequency of the corresponding <i>adar2MOt</i> was 72%.</p

Increased GluA2α^Q is sufficient to induce the neural crest defects observed in the adar2MOt.

<p>Lateral views of 18-somite stage embryos. (<b>A</b>) Expressions of crestin and sox9b in the hypo-Q/R-editing morphants are affected. Morphants created in the wild type and p53 mutant show negligible differences, indicating that the expression defects in the hypo-Q/R-editing morphants are p53-independent. (<b>B</b>) Overexpression of GluA2α^R partially restores the expressions of crestin and sox9b in the adar2MOt. Injection of gria2α^R cRNA into the wild type zygotes does not alter the expressions of crestin and sox9b (b1 and b5). However, injection of gria2α^R cRNA into the adar2MOt fully or partially rescues the expressions. Effects of rescue range from weak (b2 and b6), medium (b3 and b7) to full (b4 and b8). Weak rescue of crestin expression is defined by slightly enhanced expression in the first (1) and second (2) streams of migration neural crest cells (b2), medium rescue is defined by enhanced anterior expression (b3), and full rescue is defined by restoring the wild type expression pattern and level. Rescue of sox9b expression is classified by the enhancement of overall sox9b expression (b6-b8). Rescue efficiencies are indicated. (<b>C</b>) Overexpression of GluA2α^Q affects the expressions of crestin and sox9b. The crestin and sox9b expressions in gria2α^Q cRNA-injected embryos (c1 and c5) are affected as that of adar2MOt. The crestin and sox9b expressions are further reduced in adar2MOt by overexpressing GluA2α^Q. The additive effect varies from strong (c2 and c6), medium (c3 and c7), to weak (c4 and c8) further reduction of crestin and sox9b expressions. The occurrence rate of each phenotype is shown. 1, 2, 3, the first, second and third steams of migration cranial neural crest; e, eye; ep, epiphysis; f, forebrain; hb, hindbrain; VaNC, vagal neural crest.</p