Both p53-Dependent and -Independent Pathways Contribute to Erythroid Dysplasia in a Zebrafish Model for Diamond Blackfan Anemia.

Abstract 177

Diamond Blackfan Anemia (DBA) is a congenital autosomal dominant bone marrow failure syndrome of childhood manifested as profound anemia. The disease is characterized by enhanced sensitivity of hematopoietic progenitors to apoptosis with evidence of stressed erythropoiesis. In addition to bone marrow defects, DBA patients often have craniofacial, genitourinary, cardiac and limb abnormalities and have an increased risk of developing hematopoietic malignancies and osteosarcoma. Twenty-five percent of patients with DBA have heterozygous mutations in the ribosomal protein S19 (RPS19) gene, which encodes a component of the 40S ribosomal subunit. Additionally, a growing percentage of DBA patients lacking a mutation in the RPS19 gene have been shown to have mutations in other ribosomal protein genes. These observations support the hypothesis that DBA is a disease of altered ribosome assembly and function. It is unclear how defects in ribosomal proteins have such a specific effect on erythroid maturation and cause increased apoptosis in the erythroid compartment. An attempt to model DBA by homozygous deletion of the Rps19 gene in mice proved to be embryonic lethal, and heterozygous mice appeared to fully compensate for the loss of one Rps19 allele, in contrast to the disease observed in humans. However, two groups have successfully modeled DBA in zebrafish using an antisense morpholino (MO) approach. These studies demonstrated that similar to the human disease, rps19 deficiency leads to defective erythropoiesis, increased apoptosis and to developmental abnormalities. A central role for the tumor suppressor p53 was suggested in one of these studies. It has previously been shown that any MO injection into zebrafish embryos can lead to the activation of the p53 pathway. Therefore, in order to clarify whether p53-independent effects also contributed to the DBA phenotype in zebrafish, we utilized the p53^e7/e7 line that harbors a mutation within the p53 DNA-binding domain. Splice site and validated 5'UTR MOs targeting zebrafish rps19 were injected into one-cell stage embyros that were wildtype (WT) for p53 (AB) or mutated p53^e7/e7. Staining for hemoglobin at 48 hours post fertilization showed a profound reduction in circulating blood in both p53 wild-type and p53 mutant embryos. Although p53 mutants injected with rps19 MO show a similar reduction in hemoglobin expression to WT morphants, they have a marked improvement in their developmental defects. A 20% decrease in expression of the transcription factor GATA-1 was observed in the rps19 morphants in the p53 mutant background compared to control MO injection. The implications of this finding are being further investigated and extended to include a panel of additional erythroid-specific factors. We have observed no increase in the levels of cell death, as measured by acridine orange (AO) staining or expression of the p53-regulated apoptosis associated gene PUMA, in the p53 mutant background. Taken together, our observations indicate that the phenotype observed in DBA has both a p53-dependent and a p53-independent component. We hypothesize that the p53-dependent component of DBA is likely responsible for the increased apoptosis associated with DBA while the erythroid maturation defect is associated, in large part, with a p53-independent component. Our studies are currently focused on identifying the players in the latter pathway. These investigations should shed light on thus far undefined pathways that will likely open new avenues for drug design and development for DBA.