A Zebrafish Model of Diamond-Blackfan Anemia Results in Bone Marrow Failure and Demonstrates Defective Translation in Erythroid Cells By Ribosome Footprinting

Diamond-Blackfan anemia (DBA) is a congenital bonemarrow failure syndrome that manifests as a profoundmacrocytic anemia, which classically presents within the first year of life. Heterozygous mutations or allelic loss of one of 12 ribosomal proteins (RP) leads to apoptosis of erythroid precursors and have been identified in over 50% of DBA patients, most commonly RPS19 that accounts for 25% of all cases.

To study the role of Rps19 in erythroid development, we employed genome-editing tools to generate stable knockout line of Rps19 in zebrafish using TALENs targeting exon 1 of Rps19. We generated a stable mutant line and these mutants have a 5 bp complex indel in Exon 1 resulting in N13fs and truncated protein. Rps19 ^-/- embryos show profound developmental anomalies including a marked anemia and are embryonic lethal by 5dpf.

Rps19 ^+/- heterozygotes were indistinguishable from their WT siblings during early development and had no erythroid defect detectable by whole mount in situ hybridization (WISH) or histochemical staining. To assess the effects of Rps19 on embryonic erythropoiesis more detail, we used Rps19 mutants carrying the Tg(gata1:dsRed) and Tg(globin:eGFP) transgenes. Rps19^+/-fish were crossed and then exposed to cold stress from 12 hours post fertilization(hpf) continuously (22 degrees) and then analyzed by flow cytometry at the developmental age of 96hpf. GFP, dsRed and double GFP/dsRed expressing erythroid cells showed a reduction in cell number in Rps19^+/- compared to Rps19^+/+ siblings. Therefore, when stress is induced, the loss of one copy of rps19 is sufficient to induce anemia.

To further study the effects of Rps19 on erythroid development, we investigated the phenotype of adult fish. Adult Rps19^+/- fish at 6 months of age were significantly smaller than their siblings, weighed less and showed reduced hemoglobin levels in their peripheral blood. We then further characterized the adult rps19-deficient zebrafish phenotype by flow cytometry of whole kidney marrow using Tg(globin:eGFP);Rps19^+/-animals. Rps19^{+/ -} adults demonstrated pancytopenia, with a marked decrease in the erythroid lineage, similar to the phenotype observed in DBA patients.

Recent data have suggested that tissue specific functions of ribosomes are common and we hypothesized that such an effect may explain the tropism of Rps19^+/- phenotypes seen in DBA to erythroid and hematopoietic tissue. To test this hypothesis we assessed the effect of translation in Rps19^+/- erythroid cells compared to Rps19^+/+ using genome-wide analysis of translation by ribosome footprinting (RF) on sorted GFP+ cells from whole kidney marrow using Tg(globin:eGFP);Rps19 mutantzebrafish. In parallel we assessed RNAseq from the same samples.

RNAseq showed differential expression of genes involved in mitochondrial cell death pathways including upregulation of caspase 9 and downregulation of p53 . Stabilization of p53 and consequent p53-dependent apoptosis is a well recognized mechanism of the anemia in DBA, however our data suggest that such stabilization results in transcriptional downregulation of p53 as a compensatory mechanism to maintain a steady state level of erythropoiesis. RF data by contrast showed that the principle genes with aberrant translation in Rps19^+/- erythroid cells are other ribosomal protein genes, predominantly (although not exclusively) of the large subunit. In addition we observed that one of the most markedly upregulated footprints was on the branched chain keto acid dehydrogenase E1 subunit beta gene (encoded by bckdhb). This enzyme is part of a multimolecular complex that resides on the inner membrane of the mitochondria and is responsible for catabolism of branched chain amino acids L-Leucine and Valine, highlighting a potential underlying mechanism by which erythroid cells may be functionally leucine deficient, and thus respond to exogenous L-leucine as has previously been shown in zebrafish, murine and human cell models.

In summary, we have generated a stable Rps19 mutant line and characterized the phenotype of heterozygous Rps19^+/- mutants, which recapitulate features of DBA during developmental and adult hematopoiesis. In addition, we have used ribosome footprinting to study the effect of Rps19 heterozygosity on translation within erythroid cells of different genes, providing new insight for potential drug targets. Our on-going work is further studying the differentially expressed genes.