Rescue of the zebrafish model of congenital sideroblastic anemia.A) Whole-mount in situ hybridizations with probes for slc25a38a and slc25a38b at 24 and 34 hpf stages of development demonstrate predominant erythroid expression. For each stage and probe, head and tail views are shown and sites of expression are labeled. Abbreviations: pbi-posterior blood island, pcv-posterior cardinal vein, ss-somites, bl-blood, ret-retina, br-brain, h-heart. Expression of slc25a38b was observed in the posterior blood island, blood and posterior cardinal vein consistent with its preferential expression in erythroid progenitors and erythrocytes, whereas slc25a38a had the same blood-related expression pattern features and was also expressed in retina, brain and somites at 24 hpf. B) Representative images for hemoglobin staining using o-dianisidine of 48 hpf zebrafish embryos injected with slc25a38a+b morpholinos, or standard control morpholino (STD MO), treated with 100 mM glycine, 1 mM sodium folate, or both, starting from 4 hours post-injection. To control for morpholino specificity, we also injected 5-mismatch (5MM) versions of the same slc25a38a+b morpholinos and stained the injected embryos with o-dianisidine. For STD MO and 5MM morphants, images from only the STD MO untreated group are presented because of the low variation in o-dianisidine scores of untreated embryos versus those where glycine and/or folate were present. C) Graph of the o-dianisidine staining heme scores of 48 hpf zebrafish embryos injected with either slc25a38a+b morpholinos (slcMO) or STD MO and then treated from 4 through to 48 hpf with glycine, folate, or both glycine plus folate. All embryos were scored as having “low”, “medium” or “normal” hemoglobin levels in a blinded manner based on visual inspection of o-dianisidine staining. The total numbers (right axis) of scored embryos are indicated, from a total of three independent experiments. The “***” indicates that the p-value between the numbers of embryos in different scoring categories for slc25a38a+b morphants treated with glycine and folate versus the untreated slc25a38a+b morphant group is <0.001.