<i>m1061</i> mutants show increased <i>th</i> expression and increase in number of hypothalamic DA neurons from 3 dpf onwards.

<p>Analysis of <i>th</i> gene expression in <i>cnot8^m1061</i> mutants and WT siblings as indicated in header. (A–F) <i>th</i> transcript levels are not altered in <i>m1061/m1061</i> mutants at 1 and 2 dpf. Embryos were genotyped by PCR. (G–N) <i>m1061</i> mutants at 3 dpf display stronger WISH signal indicating increased <i>th</i> mRNA levels in DA groups 1 to 7 (arrowhead) and NA sympathetic cells (asterisk). (K, L) NA locus coeruleus (LC) and DA pretectal cells (Pre) do not display altered <i>th</i> expression levels in <i>m1061</i> mutant embryos. (M, N) The number of DA retinal amacrine cells (rAC) is reduced in <i>m1061</i> mutant embryos. Additionally <i>m1061</i> mutants display a lens defect (arrowheads). Genotypes were inferred by <i>th</i> WISH analysis. (O, P) anti-TH immunohistochemistry of <i>m1061</i> mutant embryo and WT sibling at 4 dpf document DA neurons in the ventral diencephalon. Data confirm a higher cell number of DC7 DA neurons in the caudal hypothalamus of <i>m1061</i> mutants. Dorsal view z-projections of partial confocal stacks representing the ventral diencephalon are shown. (A, B, E, F, G, H, M, N) lateral views, anterior at left; (C, D, G, H, K.L, O, P) dorsal views, anterior at left. Scale bars 100 µm in A for A, B; in C for C–F; in G for G–N; in P for O, P. Abbreviations: DC - early diencephalic DA group (DC2, DC4), 1 - ventral thalamic DA group, 2,4,5,6 posterior tubercular and hypothalamic Orthopedia-dependent DA groups, 3 - medial hypothalamic DA group, 7 - caudal hypothalamic DA group, VT - ventral thalamus, PT/H - posterior tuberculum/hypothalamus, PO - preoptic area, AAC - arch associated catecholaminergic neurons/carotid body, sym - sympathetic NA neurons, Pr - pretectum, TC - telencephalon, CH - caudal hypothalamus, LC locus coeruleus, MO - medulla oblongata, rAC - retinal amacrine cells. (Q) Quantification of CA cell numbers in <i>m1061</i> embryos at 4 dpf. Cell counts of DC1-7 and LC TH-expressing cells. Bars show the average number of CA neurons in five <i>m1061</i> mutants and five WT sibling embryos. Error bars indicate standard deviation. Significance was evaluated using Mann-Whitney test (see text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113829#pone.0113829.s002" target="_blank">Table S1</a>).</p

<i>oxtl</i>, <i>tphd2</i> and <i>crh</i> gene expression in <i>cnot8^m1061</i> embryos at 3 dpf.

<p>(A, B) <i>oxtl</i>, (C, D) <i>tphd2</i> and (E–H) <i>crh</i> WISH expression analysis in wild-type siblings and <i>cnot8^m1061</i> mutants. (A–F) lateral views. (G, H) dorsal views. Boxes indicate areas of cell counts. Sibling WT embryos developed on average 34.6 <i>crh</i> neurons and <i>cnot8^m1061</i> mutants developed on average 59.4 <i>crh</i> neurons. Significance was evaluated by Mann-Whitney test (p = 0.008). Embryos were genotyped by PCR. Scale bar 100 µm.</p

<i>cnot8^m1061</i> mutant embryos are not generally delayed in development.

<p>Gene expression analysis of <i>emx1</i>, <i>fgf8</i> and <i>krox20/egr2b</i> in <i>cnot8^m1061</i> mutants and wild-type siblings at 1, 2 and 3 dpf. (A–F) <i>emx1</i>, (G–L) <i>fgf8</i> and (M–R) <i>krox20/egr2b</i>. Embryos were genotyped by PCR. All pictures show lateral views. Scale bars 100 µm.</p

<i>sim1a</i>, <i>otpa and nkx2.1a</i> expression in <i>cnot8^m1061</i> embryos and WT siblings.

<p>Analysis of <i>otpa</i> (A–D), <i>sim1a</i> (E, F) and <i>nkx2.1a</i> (G–J) gene expression in <i>cnot8^m1061</i> mutant embryos and wild-type siblings, stages as indicated. (A–F, I, J) lateral views. (G, H) dorsal views. Embryos were genotyped by PCR. Scale bar 100 µm.</p

<i>fgf3/lia</i> FGF signaling mediates the increase in caudal hypothalamic DC7 DA neurons in <i>cnot8^m1061</i> mutant embryos.

<p>(A–F) Analysis of DA neurons in <i>cnot8^m1061</i> mutant embryos combined with a mutation in the <i>fgf3</i> locus (<i>lia^t24152</i>) or pharmacological suppression of FGF signaling by SU5402. Embryos were fixed at 4 dpf, stained by anti TH immunofluorescence and DA neurons documented by confocal microscopy. Shown are Z-projections of confocal stacks representing the ventral diencephalic DA groups 1 to 7 (dorsal views). Scale bar 50 µm. (A–D) DA neurons in WT, <i>cnot8^m1061</i> or <i>lia^t24152</i> single mutant, and <i>cnot8^m1061</i>, <i>lia^t24152</i> double mutant embryos. Double mutant embryos show loss of <i>cnot8^m1061</i>-mediated increase of DC7 caudal hypothalamic DA neurons. (E, F) DA neurons in WT and <i>cnot8^m1061</i> mutant embryos treated with SU5402 from 42 to 48 hpf. Inhibition of FGF signaling by SU5402 reduces the number of DC7 caudal hypothalamic DA neurons in <i>cnot8^m1061</i> mutants below WT levels. (G) Quantification of effects on CA neurons by cell counting of forebrain DA neuronal clusters and locus coeruleus NA neurons in genetic and experimental conditions as indicated in the index at right. Each bar shows the average number of CA neurons in five independent embryos for each experimental condition. Error bars indicate standard deviation. Average DC7 cell numbers: <i>cnot8+/+ lia +/+</i> 50.8 (WT control); <i>cnot8-/- lia +/+</i> 103.2; <i>cnot8-/- lia -/-</i> 52.6; <i>cnot8+/+ lia -/-</i> (38.8); <i>cnot8+/+</i> SU5402 20.6; <i>cnot8-/-</i>SU5402 56.2. Significance was evaluated by Mann-Whitney test. The cell count in single mutant <i>cnot8^m1061</i> (-/-)<i>lia^t24152</i> (+/+) is significantly different from single mutant <i>cnot8^m1061</i> (+/+) <i>lia^t24152</i> (-/-) embryos (p = 0.008). Comparison of <i>cnot8^m1061</i>, <i>lia^t24152</i> double mutant and WT embryos reveal no significant difference (p = 0.45). For SU5402 treatments, the number of DC7 DA neurons differs significantly between WT controls and SU5402 treated WT (p = 0.008) and between <i>cnot8^m1061</i> and SU5402-treated <i>cnot8^m1061</i> embryos (p = 0.008). For all other catecholaminergic groups, no significant differences were observed when WT was compared to single mutants, double mutants, or SU5402-treated embryos.</p

The <i>cnot8^m1061</i> mutation affects FGF receptor expression levels.

<p>The expression of the four zebrafish FGF receptor genes <i>fgfr1</i>, <i>2</i>, <i>3</i> and <i>4</i> was analyzed by WISH in <i>cnot8^m1061</i> mutants and WT siblings at 1, 2 and 3 dpf. All pictures show lateral views. Embryos were genotyped by PCR. Scale bar 100 µm in A for A–X.</p

The live phenotype and positional cloning of the <i>cnot8^m1061</i> mutation.

<p>(A) Comparison of <i>cnot8^m1061</i> mutant to wild-type sibling embryos at 5 dpf; <i>cnot8^m1061</i> mutants display edema formation in the eyes and brain (top row, arrowheads) as well as cardiac and yolk sac (bottom row, arrowhead). Top row dorsal view, bottom row lateral view, anterior to the left. Scale bar 100 µm. (B) Scheme of the <i>m1061</i> genetic interval. Gene and marker positions were obtained from Ensembl Zv8. CR855270.17 p9 and BX927237 p4 define the interval which comprises the genes <i>zgc:77151</i>, <i>MRP L22</i>, <i>gemin5</i>, <i>cnot8</i>, <i>kibra/WWC1</i>, <i>ARL10</i> and <i>Nop16</i> (data not shown). Proximal SSLP markers are highlighted in blue. Distal SSLP markers are highlighted in brown. Numbers provided with SSLP markers reflect recombination events identified per number of meioses analyzed. (C) Sequencing of genomic DNA amplified by PCR from individual <i>m1061</i> homozygous WT and mutant embryos. The C-to-T mutation at bp 82 of the ORF results in the formation of a premature stop codon in <i>m1061</i> mutant <i>cnot8</i> ORF. Numbers (79–87) indicate ORF bp position. (D) Zebrafish Cnot8 comprises 286 amino acids and contains an RNAse D domain which is truncated in <i>cnot8^m1061</i> mutants. (E–J) Expression analysis by WISH using <i>cnot8</i> antisense probes. Sense controls processed exactly in parallel with the antisense reactions are shown as insets in E, F, G and H to evaluate background stain intensities. (E) Maternal mRNA is detected at 4 cell stage (dorsal view). (F) Ubiquitous expression of <i>cnot8</i> mRNA at 4 hpf (lateral view). (G, H, I) <i>cnot8</i> continues to be expressed ubiquitously at 50% epiboly, 1, 2 and 3 dpf (lateral views). Scale bars 100 µm.</p

<i>fgf3</i> gene expression is increased in <i>cnot8^m1061</i> mutants.

<p>Analysis of <i>fgf3</i> gene expression in <i>cnot8^m1061</i> mutants and WT siblings 2 dpf (A, B), and 3 dpf (C, D). All pictures show lateral views. Embryos were genotyped by PCR. Scale bar 100 µm.</p

<i>erm</i> and <i>pea3</i> expression in <i>cnot8^m1061</i> mutants and WT siblings.

<p>(A, B) <i>erm</i> expression in WT siblings and <i>cnot8^m1061</i> mutants at 3 dpf. (C–H) <i>pea3</i> expression in WT siblings and <i>cnot8^m1061</i> mutants at (C, D) 3 dpf and (E–H) 2 dpf. (E–G) <i>cnot8^m1061</i> mutants show higher <i>pea3</i> mRNA levels in cells surrounding the lens in comparison to WT siblings (arrowheads). (A–D, G, H) lateral views. (E, F) dorsal views, anterior at left. Embryos were genotyped by PCR. Scale bars 100 µm in B for (A, B), in D for (C, D) and in H for (E–H).</p