Fig. 3

The topbp1 gene is disrupted in mutant^cas003.

(A) Positional cloning of mutant^cas003. 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 topbp1, tmem108, cdv3 and vps41. 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 topbp1 was sequenced in the wild-type sibling (top) and mutant^cas003 (bottom). There is a C to T mutation in the mutant^cas003 embryos, which is a nonsense mutation. (C) Comparison of vertebrate TopBP1 and zebrafish TopBP1^cas003. As human TopBP1 (Hs. TopBP1) and mice TopBP1 (Mm. TopBP1), zebrafish TopBP1^WT (Dr. TopBP1) contains eight BRCT domains, while the eighth BRCT domain and putative nuclear localization signal (NLS) is missing in TopBP1^cas003 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^cas003. (E-G′) Topbp1 morphants can phenocopy topbp1^cas003. (E) Topbp1 MO can block the translation of topbp1 mRNA. The topbp1 MO, as validated in S3 Fig, was injected into one-cell stage wild-type embryos to produce topbp1 morphants. (F-G′) WISH results of c-myb in the control morphants (F, F′) and topbp1 morphants (G, G′) at 3dpf. topbp1 morphants show decreased c-myb 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. topbp1^WT and topbp1^cas003 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 ubi: topbp1^WT and ubi: topbp1^cas003 respectively. (I-L) WISH analysis with c-myb probe in the CHT region (at 5dpf) of sibling, topbp1^cas003 mutant and topbp1^cas003 mutant with transient transgenesis of ubi: topbp1^WT or ubi: topbp1^cas003. 28 embryos out of 45 topbp1^cas003 mutants were rescued by topbp1^WT, but none by topbp1^cas003 (n = 14).