The <i>topbp1</i> gene is disrupted in mutant^{<i>cas003</i>}.

<p>(A) Positional cloning of mutant^{<b><i>cas003</i></b>}. Bulk segregation analysis (BSA) revealed that the mutation occurred on chromosome 24 (Chr. 24). After high-resolution mapping with SSLPs, the point mutation was flanked by SSLP markers L0310_5 (1 recombinant out of 2082 meiosis) and R0310_4 (5 recombinants out of 2082 meiosis). This region contains four genes including <i>topbp1</i>, <i>tmem108</i>, <i>cdv3</i> and <i>vps41</i>. The blue line represents Chr. 24; the positions and the recombinations of the SSLP markers on Chr. 24 or BACs are indicated. The SSLP markers which are on the same side of the mutation site are shown in the same color. (B) The coding region of <i>topbp1</i> was sequenced in the wild-type sibling (top) and mutant^{<b><i>cas003</i></b>} (bottom). There is a C to T mutation in the mutant^{<b><i>cas003</i></b>} embryos, which is a nonsense mutation. (C) Comparison of vertebrate TopBP1 and zebrafish TopBP1^{<b><i>cas003</i></b>}. As human TopBP1 (Hs. TopBP1) and mice TopBP1 (Mm. TopBP1), zebrafish TopBP1^{<b><i>WT</i></b>} (Dr. TopBP1) contains eight BRCT domains, while the eighth BRCT domain and putative nuclear localization signal (NLS) is missing in TopBP1^{<b><i>cas003</i></b>} due to the nonsense mutation (*). The red and pink BRCT domains were predicted by SMART software. The orange BRCT domains were reported previously. The molecular sizes of the protein are indicated in the right side. (D) Western blotting analysis showing reduced protein size of TopBP1^{<b><i>cas003</i></b>}. (E-G’) <i>Topbp1</i> morphants can phenocopy <i>topbp1</i>^{<b><i>cas003</i></b>}. (E) <i>Topbp1</i> MO can block the translation of <i>topbp1</i> mRNA. The <i>topbp1</i> MO, as validated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005346#pgen.1005346.s003" target="_blank">S3 Fig</a>, was injected into one-cell stage wild-type embryos to produce <i>topbp1</i> morphants. (F-G’) WISH results of <i>c-myb</i> in the control morphants (F, F’) and <i>topbp1</i> morphants (G, G’) at 3dpf. <i>topbp1</i> morphants show decreased <i>c-myb</i> expression. (F’, G’) The enlarged views of the dotted rectangle region in the left columns. The penetrance of the indicated phenotype is shown in the bottom left of each panel. (H) Construction of the plasmids used in Tol2-transposease-mediated rescue assays. <i>topbp1</i>^{<b><i>WT</i></b>} and <i>topbp1</i>^{<b><i>cas003</i></b>} driven by the ubiquitin promoter, followed by the P2A peptide and the mCherry coding sequence, are cloned into Tol2 transposon vector. These constructs are abbreviated as <i>ubi</i>: <i>topbp1</i>^{<b><i>WT</i></b>} and <i>ubi</i>: <i>topbp1</i>^{<b><i>cas003</i></b>} respectively. (I-L) WISH analysis with <i>c-myb</i> probe in the CHT region (at 5dpf) of sibling, <i>topbp1</i>^{<b><i>cas003</i></b>} mutant and <i>topbp1</i>^{<b><i>cas003</i></b>} mutant with transient transgenesis of <i>ubi</i>: <i>topbp1</i>^{<b><i>WT</i></b>} or <i>ubi</i>: <i>topbp1</i>^{<b><i>cas003</i></b>}. 28 embryos out of 45 <i>topbp1</i>^{<b><i>cas003</i></b>} mutants were rescued by <i>topbp1</i>^{<b><i>WT</i></b>}, but none by <i>topbp1</i>^{<b><i>cas003</i></b>} (n = 14).