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.