Expression of <i>Slc34a2a</i> and related transcripts during zebrafish embryogenesis.

<p>(A) Schematic representation of the <i>Slc34a2a</i>, <i>Slc34a2a</i>(as) and <i>Rbpja</i> loci. The antisense transcript <i>Slc34a2a</i>(as) is depicted in red. (B) RT-qPCR analysis of <i>Slc34a2a</i>, <i>Slc34a2a</i>(as) and <i>Rbpja</i> transcripts including the paralog <i>Slc34a2b</i>. Based on negative controls using RNA as an input, the detection limit was set at a ΔCt of 12 which is in agreement with ISH results. (C) Demonstration of <i>Slc34a2a</i>, <i>Slc34a2a</i>(as), <i>Rbpja</i>, <i>Slc34a2b</i> and <i>Shh</i> (Sonic Hedgehog) transcripts at progressing stages of development by whole mount ISH.</p

Injection of <i>Slc34a2a</i> RNA into fertilized zebrafish eggs.

<p>A) Visual classification of malformations depending on the severity of the defect: Level 1, wild type; level 2, one organ affected (size, shape or function, e.g. heart rate); level 3, 2–3 organs affected, level 4, multiple malformations; level 5, developmental arrest. B) Phenotypic characterization of zebrafish embryos injected with various RNAs, <i>Slc34a2a</i> (554 embryos in total), antisense (94 embryos) and <i>Slc34a2b</i> (538 embryos). C) (i) Anatomy of a 48 hpf zebrafish embryo: Y, yolk sac; E, eye; O, otic vesicle (ear); R1/R7, rhombomeres; M, mesencephalon; C, cerebellum, in red. (ii) non injected wild type embryo; (iii) <i>Slc34a2a</i> injected embryo; (iv) wild type embryo, Eng2 stained; (v) <i>Slc34a2a</i> injected embryo, Eng2 stained; (vi, vii) <i>Slc34a2a</i>(as) and <i>Slc34a2b</i> Eng2 stained.</p

Injection of non- protein coding <i>Slc34a2a</i> RNA and <i>Slc34a2a</i> fragments interfere with zebrafish development.

<p>A) Schematic representation of a zebrafish head at 48 hpf; forebrain, blue; eyes, yellow; otic vesicles, green and cerebellum, red. Middle and left, wild type and <i>Slc34a2a</i>-FS injected embryo, respectively. Red arrows indicate the position of the cerebellum. B) Phenotypic quantification of <i>Slc34a2a</i> and <i>Slc34a2a</i>-FS injected embryos (364 <i>Slc34a2a</i>-FS injected embryos were assessed). C) Schematic representation of the fragments generated, even numbers represent sense orientation; uneven numbers, antisense orientation. The large black boxes represent exons comprised in the relevant fragments, the open boxes are exons that are not represented in the injected fragments. The small boxes in red indicate potential sites of hybridization of the injected fragments with an endogenous transcript on the opposite strand. D) Top view of 48 hpf embryos with the fragments (Frag) injected as indicated. E) Phenotypic assessment of injected embryos (90 or more per RNA). F) Eng2 stained embryos injected with the indicated fragments and the relevant controls. All the embryos were tested in parallel with the same solutions and under identical conditions to allow for a comparison of the relative intensities.</p

Morpholino knockdown of <i>Slc34a2a</i>(as) and Dicer.

<p>A) RT-qPCR quantification of <i>Slc34a2a</i>, <i>Slc34a2a</i>(as) and <i>Slc34a2b</i> after splice site morpholino injection at 24 hpf. Wild type non injected controls, light blue bars; 5 ng splice-site MO injected embryos are in dark blue; 5 ng scrambled MO injected embryos are in blue. B) Phenotypic characterization of MO injected embryos at 24 and 48 hpf. The injected oligonucleotides and quantities are indicated below the bars. Phenotypic scaling was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178219#pone.0178219.g002" target="_blank">Fig 2</a>. C) Rescue of cerebellum development by Dicer knockdown. Phenotypic assessment of embryos injected with combinations of Dicer MO, p53 MO and <i>Slc34a2a</i>. D) 48 hpf zebrafish embryos injected with Dicer MO, p53 MO and <i>Slc34a2a</i> as indicated in the pictures. In the upper panel, heads with red arrows indicating the cerebellum are shown; the lower panel shows ISH of embryos with an Eng2 probe.</p

Ectopically expressed <i>Slc34a2a</i> sense-antisense transcripts cause a cerebellar phenotype in zebrafish embryos depending on RNA complementarity and Dicer

<div><p>Natural antisense transcripts (NATs) are complementary to protein coding genes and potentially regulate their expression. Despite widespread occurrence of NATs in the genomes of higher eukaryotes, their biological role and mechanism of action is poorly understood. Zebrafish embryos offer a unique model system to study sense-antisense transcript interplay at whole organism level. Here, we investigate putative antisense transcript-mediated mechanisms by ectopically co-expressing the complementary transcripts during early zebrafish development. In zebrafish the gene <i>Slc34a2a</i> (Na-phosphate transporter) is bi-directionally transcribed, the NAT predominantly during early development up to 48 hours after fertilization. Declining levels of the NAT, <i>Slc34a2a</i>(as), coincide with an increase of the sense transcript. At that time, sense and antisense transcripts co-localize in the endoderm at near equal amounts. Ectopic expression of the sense transcript during embryogenesis leads to specific failure to develop a cerebellum. The defect is RNA-mediated and dependent on sense-antisense complementarity. Overexpression of a <i>Slc34a2a</i> paralogue (Slc34a2b) or the NAT itself had no phenotypic consequences. Knockdown of Dicer rescued the brain defect suggesting that RNA interference is required to mediate the phenotype. Our results corroborate previous reports of <i>Slc34a2a</i>-related endo-siRNAs in two days old zebrafish embryos and emphasize the importance of coordinated expression of sense-antisense transcripts. Our findings suggest that RNAi is involved in gene regulation by certain natural antisense RNAs.</p></div

Injection of <i>Slc34a2a</i> RNA and related constructs into fertilized zebrafish eggs.

<p>A) ISH of wild type and injected embryos at 24 hpf. Horizontal labels at the top indicate the injected material, vertical labels, left, represent the probes used for ISH. B) RT-qPCR of injected zebrafish embryos; Slc34a2a, Slc34a2a(as) and Slc34a2b RNA was injected as indicated with the different colour from brown to blue and assayed after 10 and 24 hpf. The left group represents RT-qPCR reactions with Slc34a2a-specific primers; the middle group with Slc34a2a(as)-specific primers and the right group with Slc34a2b-specific primers. The values for non-injected controls are indicated with grey, transparent boxed.</p

Conditional expression of <i>RockDN</i> in NCC.

<p><b>A</b>) Strategy for expressing <i>RockDN</i> construct in NCC. <b>B</b>) Quantitative real time PCR for the <i>CAT</i> gene cassette, using RNA extracted from pharyngeal arches 1 and 2, from E11.5 mutant <i>RockDN⁺;Wnt1-cre⁺</i> and control embryos. There is a statistically significant (P<0.05 *), 13 fold decrease, in the expression of the <i>CAT</i> box in the mutant sample, as calculated using the one-way Anova test.</p

Disruption of the actin cytoskeleton and vinculin-containing focal contacts in E9.5 <i>RockDN;Wnt1-cre</i> embryos.

<p>A,B,E,F,I,J,M,N (line i in Q) are sections from first pharyngeal arch and C,D,G,H,K,L,O,P (line ii in Q) are from the frontonasal processes. A–H show phalloidin (red) and caspase 3 (green) immunofluorescence, with E–H being magnified regions as shown by the boxes in A–D, respectively. I–L show vinculin (red) and caspase 3 (green) dual immunofluorescence, with M–P being magnified regions as shown by boxes in I–L. The dotted lines in C,D,G,H,K,L,O,P indicate the boundary between the inner ectomesenchyme and the neural ectoderm and the surface ectoderm. <b>A–H</b>) Filamentous actin, labelled with phalloidin (red) outlines the cells in NCC-derived ectomesenchyme and neural ectoderm in the first pharyngeal arch (A,E) and frontonasal processes (C,G) of control embryos. Cortical phalloidin staining is lost in the ectomesenchyme from <i>RockDN;Wnt1-cre</i> mutants (F,H) but is maintained in the neural ectoderm (compare G with H). In addition, intense phalloidin-labelled foci are observed throughout the ectomesenchyme of the <i>RockDN;Wnt1-cre</i> mutants (dense red foci, blue arrow in F,H). Green caspase 3-positive cells are interspersed (white arrow) and overlapping with the phalloidin-intense cells (arrowheads in F,H). <b>I–L</b>) Vinculin and caspase 3 staining. In the pharyngeal arch the vinculin staining is not restricted to the centre of the arch in the mutant (compare J with I). In the frontonasal processes vinculin outlines the boundary between the surface ectoderm and the neural ectoderm with the inner NCC-derived ectomesenchyme (dotted lines in K,L). This discrete vinculin staining is lost in the <i>RockDN;Wnt1-cre</i> mutants (compare P with O). cas3 = activated caspase-3; mes = mesenchyme; ne = neural ectoderm; phall = phalloidin; se = surface ectoderm; vin = vinculin. Scale bar = 50 µm.</p

Abnormalities in formation of the craniofacial bones in <i>RockDN;Wnt1-cre</i> embryos.

<p><b>A,B</b>) In severely affected <i>RockDN;Wnt1-cre</i> embryos at E14.5, the frontonasal bones (stained with alcian blue) are absent (arrow in B, compare to A). Meckel's cartilage is also reduced in size (arrowhead in B, compare with A). <b>C,D</b>) Bone (red) and cartilage (blue) staining of a mildly affected <i>RockDN;Wnt1-cre</i> embryo at E18.5 (D), shows that the maxilla (arrowhead) and mandibular (arrow) bones are well formed, although the hyoid bone (red arrow) is reduced in size in mutant embryos. <b>E,F</b>) Inferior views of the base of the skull in mildly affected embryos shows that the basisphenoid and the presphenoid bones are hypoplastic in <i>RockDN;Wnt1-cre</i> embryos, whereas the nasal septum is completely missing. Moreover, the maxillary bones are widely separated in mutant embryos (double arrow in F), compared to control littermates (E). bs = basisphenoid; n = nasal septum; ps = presphenoid. Scale bar = 500 µm.</p

Disruption of focal adhesions and extracellular matrix in E9.5 <i>RockDN;Wnt1-cre</i> embryos.

<p>A,B,E,F are sections of the first pharyngeal arch (line i in O) and C,D,G,H,I–N are from the frontonasal processes (line ii in O). <b>A–H</b>) E–H are magnified regions shown in the boxes on A–D, respectively. Paxillin has a cortical distribution in the first pharyngeal arch in the control embryos (A,E) and also marks the boundary between the neural ectoderm and NCC-derived ectomesenchyme (arrow in C,G) in the frontonasal processes. This boundary staining is lost in the mutant (arrow in D,H) and there are intense paxillin positive foci in the pharyngeal arch and frontonasal processes (arrowheads in B,F,D,H), confirming loss of cell-substrate adhesion. <b>I–L</b>) Laminin was lost from the ectomesenchyme-neural ectoderm boundary (compare arrowhead in J with L) and was abnormally distributed in the surface ectoderm in the frontonasal processes in the <i>RockDN;Wnt1-cre</i> embryos (compare arrow in I with K). The H&E staining of the same sections are shown in M and N, allowing visualisation of the different cellular layers. fnp = frontonasal process; pa1 = first pharyngeal arch; ne = neural ectoderm; se = surface ectoderm; mes = mesenchyme. Scale bar in A–D,I,K = 50 µm; M,N = 40 µm.</p