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 e 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.
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