We assessed levels of cell death

We assessed levels of cell death using annexin V and propidium iodide staining. The tp53 animals showed a 20% increase in erythroid cell death when assessed by propidium iodide staining (p = 0.02, Figures 4A and S3A), but no increase in annexin V staining (Figures S3A and S3B), similar to what was observed with UV irradiation exposure in tp53 animals (Berghmans et al., 2005). These observations suggest that perhaps non-apoptotic pathways were at work, such as necrosis/necroptosis, as previously described (Galluzzi and Kroemer, 2008). When we assessed the phenotype of tp53 homozygotes after pro-oxidant exposure, we found a severe reduction in hemoglobin-producing harmine shown by o-dianisidine staining. This resulted in a significant amount of edema and increased mortality, especially at higher concentrations of pro-oxidant exposure (Figures 4B and 4C, p < 0.0001). This phenotype was similar to observations we have seen in G6PD-deficient zebrafish under elevated oxidative stress (Patrinostro et al., 2013). Incidentally, pro-oxidant-stressed tp53 mutant zebrafish showed a partial rescue in phenotype when co-exposed with necrostatin, a potent inhibitor of receptor-interacting protein kinase-1 (RIPK1), a central mediator of necroptosis (Degterev et al., 2008), although a more thorough interrogation of necroptotic pathways, such RIP3, would be definitive (Figure S3C). Being that TP53 controls many intracellular pathways with a wide variety of biological functions, we took an agnostic experimental approach and performed RNA-seq on tp53 homozygotes under oxidative stress and wild-type zebrafish to get an idea of which major cellular processes differ between them. We found 753 genes that were differentially expressed by greater than a 2-fold variance (Figure 4D, top genes shown in Table S2). We performed a gene ontology (GO) term analysis for gene enrichment and found the largest biological processes affected in tp53 homozygotes were “metabolic processes” (Figure 4E). Mitochondria are the central regulators of cellular metabolism and account for a majority of the cellular ATP production (McBride et al., 2006). Due to the optical clarity of the zebrafish, they offer unparalleled visualization of organelles when using fluorescent organelle-targeted reporters. Therefore, to determine if there was any dysregulation in mitochondria shape or number, we crossed Gata1:DsRed and Gata1:DsRed/tp53 animals to mito:GFP transgenic animals, which have a mitochondrial localization signal sequence fused to GFP, allowing for microscopic evaluation of mitochondrial size, shape, and enumeration by flow cytometry (Kim et al., 2008); the resultant offspring displayed DsRed-positive Gata1+ erythroid cells with GFP-tagged mitochondria (Figure 4F). We noticed no differences in Gata1+ erythroid cell mitochondria size or shape in tp53 compared with tp53 animals (not shown). There was also no difference in Gata1+ erythroid cell mitochondrial content between tp53 and tp53 animals (Figure 4G). Although there was no effect of mutant tp53 on mitochondrial content, we hypothesized that mitochondrial function could be altered. Therefore, we interrogated total mitochondrial respiration using a Seahorse biochemical analyzer. We compared the oxidative phosphorylation (OXPHOS) metabolic profile of wild-type animals with that of tp53 animals and found that tp53 mutants had a significantly elevated basal oxygen consumption rate (OCR) compared with that of wild-type (Figures 4H–4J and S3D). Due to their elevated basal OCR, the tp53 mutant animals were severely deficient in metabolic reserve capacity (p < 0.0001, Figure 4J). Although we can only measure OCR from the total animal and not specifically in erythroid precursors, we believe these findings translate to multiple cell types. We next utilized the ability of oligomycin, an inhibitor of H+-ATP-synthase, to reduce ATP, oxidative phosphorylation, and ROS production (Shchepina et al., 2002). We found that oligomycin treatment of pro-oxidant-exposed tp53 mutants led to a partial rescue in edematous phenotype (p < 0.0007, Figure 4K), highlighting the significant role that mitochondria play in ROS-mediated erythroid cell death.