Phenotype of the zebrafish bloody minded (blm) mutant. A) Mutants can be first recognized at ~27 hpf when signs of malformation in the head are visible (red arrows). At 2 and 3 dpf, eyes are much smaller and the tail is bent dorsally. B) Plastic sections through the eye reveal apoptotic cells in the retina (red arrow) and in the brain at 27 hpf. There is no proper lamination of the retina and necrosis proceeds. Abbreviations: hpf - hours post fertilisation, dpf - days post fertilisation, R - retina, L - lens, PhL - photoreceptor layer, INL - inner nuclear layer, IPL - inner plexiform layer, GCL - ganglion cells layer.

top2a mutants exhibit defects in cell cycle progression. A) Flow cytometry analysis of larvae at 27 hpf (red plot - wt, blue - blm) reveals an increased fraction of cells in G2/M phase in blm mutants. B) Representative Z-series projections showing that the total number of mitotic cells (stained with PH3 antibody - red) is lower in the eye of 27 hpf blm mutants (n = 58) than in wild type larvae (n = 70). C) The percentage of mitotic cells (PH3 positive) relative to the total number of DAPI stained cell nuclei is significantly (p = 0.036) higher in blm mutants compared to wild type as assessed from n ≥ 23 sections through the head and spinal cord of the N = 5 mutant and wild type larvae. D) Real time PCR of cell cycle markers p21-like (G1 phase) and ccnb1 (G2/M phase) in wildtype and blm larvae at 27 hpf shows no significant difference in expression levels (p = 0.21 and 0.38, respectively). Expression of atoh7, a marker of retinogenesis, is also equivalent in eyes of blm and wildtype siblings. Presented data are average of 3 replicate experiments, each comprising pools of 16-35 larvae. Error bars represent the standard error of the mean (C-D). E) Acridine orange staining of apoptotic cells in wildtype and blm larvae at 24 hpf. Arrows point to regions of increased apoptosis in blm mutants.

Overlapping expression, but functional divergence of top2a and top2b paralogues in vivo. A) RT-PCR shows that both top2a and top2b are expressed in the eye at 2, 3, 4 and 5 dpf. B-G) Wholemount in situ hybridization reveals that top2b is expressed in the anterior of wildtype larvae including the forebrain, midbrain and eye (B-D) at 22 hpf and in the forebrain, midbrain, branchial arches and retina (F-G) at 3 dpf. E is the negative control sense probe; D and G are sections through the eye. H) RT-PCR at 3 dpf shows similar abundant expression of top2b in the eye and body of blm mutants and wildtype sibling. I-L) Wholemount in situ hybridization reveals a similar spatial expression pattern of top2b in blm (I, K) and wildtype larvae (J, L) at 3 dpf. M) top2b expression levels at 33 hpf following injection of top2b mRNA into 1-2 cell stage offspring of blm carriers. blm larvae overexpressing top2b RNA by 2.7-5.9 fold show no evidence of phenotypic rescue. N = 3 replicate experiments, n = 50 uninjected wild type (wt cont), n = 29 top2b RNA injected wild type (wt inj), n = 13 uninjected blm (blm cont), n = 17 top2b RNA injected blm (blm inj), n = 6 top2b RNA injected malformed. N) Phenotypes recorded at 33 hpf following injecting offspring of carriers of blm mutation with zebrafish top2b mRNA (N ≥ 3 replicate experiments, n = 117 uninjected wild type, n = 40 uninjected blm, n = 29 top2b RNA injected wild type, n = 17 top2b RNA injected blm, n = 6 top2b RNA injected malformed). Error bars represent the standard error of the mean. Abbreviations: ba - branchial arches, e - eye, fb - forebrain, hb - hindbrain, ln - lense, mb - midbrain, r - retina.

Chemical inhibition of Top2a disrupts pre-MZT development of zebrafish embryos. A) Schematic of the experimental procedure. 1-2 cell stage embryos were treated with Top2a inhibitors for 1.5 hours and their developmental progression was analysed. B-C) Graphs representing the percentage of embryos at a range of developmental stages following treatment with ICRF-193 (B) or etoposide (C). Top2a inhibition changes the staging distribution of embryos which peak at 16-32 cell stage in vehicle controls (DMSO) to peaking at the 8-16 cell stage in drug treated populations. The "unclassified" category includes embryos that do not fit into the standard developmental morphologies, ones in which all cells do not have typical optical transparency, and ones that are malformed. D) Table grouping the percentage of embryos at ≤ 16 cell stage or ≥ 32 cell stage for each drug treatment. Top2a inhibition increases the number of embryos ≤ 16 cell stage by up to 26%, and decreases the number of embryos ≥ 32 cell stage by up to 35%. Asterisks represent statistically significant differences (p < 0.005) to DMSO controls.

Model depicting how maternal top2a enables pre-MZT development of zebrafish embryos and post-MZT development of top2a mutants.

blm arises from a mutation in the top2a gene. A) Mapping of blm reveals that the gene is located on chromosome 12. B) top2a was selected as a candidate gene and sequencing of mutant cDNA shows a point mutation (A→T) in Lys residue 335 resulting in a premature stop codon. C) A schematic of wildtype Top2a protein and the truncated proteins in hi3635, blm and can4 mutants D) Complementation assay performed to confirm that blm is top2a mutation. blm heterozygotes were crossed to hi3635 carriers and the resulting hybrid offspring present with blm phenotype.

top2a, but not top2b, is expressed pre-MZT. RT-PCR of wt embryos (A-B) and real time PCR of offspring from blm carriers (C-D) shows that maternal top2a (A, C), but not top2b (B, D), is present pre-MZT at 8- and 16-cell stages. top2a transcript levels decrease in blm siblings from 27 hpf to 3 dpf, and the expression in blm mutants is always significantly lower (C). The levels of top2b transcript also decrease in wildtype siblings from 27 hpf to 3 dpf, and although top2b levels in blm mutants and siblings is similar at 27 hpf, top2b is dramatically reduced in the mutants by 2 dpf (D). Error bars represent the standard error of the mean. At 8-cell, 16-cell, 4 hpf and 10 hpf stages mutant and wild type offspring are pooled. In graphs light blue (C) and light green bars (D) represent pooled wiltype and mutant embryos; dark blue/green bars represent blm mutants and white bars represent wildtype larvae.

Chemical inhibition of Top2a pre-MZT does not affect cell cycle profile at 27 hpf. A) Schematic of the experimental procedure. 1-2 cell stage embryos were treated with 100 μM ICRF-193 for 3.5 hours when the drug was removed and the cell cycle profile analysed at 27 hpf. B-C) Flow cytometry analysis of cell cycle profiles of wt (B) and mutant (C) embryos. D) Table documenting the percentage of cells in cell cycle phases following ICRF-193 treatment.

Inhibition of maternal Top2a in pre-MBT cycling extracts delays cell cycle progression by lengthening the M-phase. A) Cycling extracts from activated Xenopus eggs were treated with 20 μM Top2a inhibitor ICRF-193 diluted in 1% DMSO. To monitor cell cycle progression samples were taken every 10 minutes and phospho-histone H3 expression relative to GAPDH expression plotted. Blue and red bars indicate M phase in DMSO control and ICRF-193 treated extracts, respectively. B) Western blot analysis of phospho-Histone H3 and GAPDH expression levels. C) The increase in M phase length in two different cycling extracts treated with ICRF-193. M phase lengthened on average by 40% (SD = 3.14).