Hematopoietic defects in rps29 mutant zebrafish depend upon p53 activation

Abstract

Disruption of ribosomal proteins is associated with hematopoietic phenotypes in cell culture and animal models. Mutations in ribosomal proteins are seen in patients with Diamond Blackfan anemia (DBA), a rare congenital disease characterized by red cell aplasia and distinctive craniofacial anomalies. A zebrafish screen uncovered decreased hematopoietic stem cells (HSCs) in embryos with mutations in ribosomal protein rps29. Here, we determined that rps29-/- embryos also have red blood cell defects and increased apoptosis in the head. As the p53 pathway has been shown to play a role in other ribosomal protein mutants, we studied the genetic relationship of rps29 and p53. Transcriptional profiling revealed that genes up-regulated in the rps29 mutant are enriched for genes up-regulated by p53 after irradiation. p53 mutation near completely rescues the rps29 morphological and hematopoietic phenotypes, demonstrating that p53 mediates the effects of rps29 knockdown. We also identified neuronal gene orthopedia protein a (otpa) as one whose expression correlates with rps29 expression, suggesting that levels of expression of some genes are dependent on rps29 levels. Together, our studies demonstrate a role of p53 in mediating the cellular defects associated with rps29 and establish a role for rps29 and p53 in HSCs and red blood cell development.

Whole-exome sequencing and functional studies identify RPS29 as a novel gene mutated in multicase Diamond-Blackfan anemia families

“…39 These include decreased hematopoietic stem cell markers, defects in RBC development, and an increase in apoptotic cells. 12,39 Here we show that both our DBA-associated RPS29 mutations failed to rescue the defective Hb levels in the rps29 2/2 zebra fish compared with WT human RPS29.…”

“…12 Similarly, the rps19 zebra fish DBA model and mouse models also show that ribosomal haploinsufficiency results in p53 activation that leads to the defective erythropoiesis underlying the DBA-associated anemia. 40,41 Together with the DBAassociated RPL5, RPL11, RPS7, RPL26, and RPS19 genes that have been shown to induce p53, RPS29 is an additional DBA gene linked to p53 activity.…”

“…In addition, morpholinos can be used to transiently knock down genes in the embryo, and in vivo overexpression can be performed by injecting DNA or RNA. 12 Here, we employed whole-exome sequencing (WES) to survey the exome for novel DBA disease-causing genes and used zebra fish as a model system to determine the consequences of the DBA-associated mutation. WES was performed on 13 individuals with DBA who lacked a mutation in any of the known DBA genes and family members from 2 large families with multiple DBA-affected individuals from the longitudinal IBMFS cohort at the National Cancer Institute (NCI).…”

Hooked! Modeling human disease in zebrafish

“…Other anemias have been phenocopied using morpholino-mediated knockdown (34, 35), including diamond blackfan anemia (DBA), which is modeled by knockdown of ribosomal protein RSP19 (36). Characterization of the RSP19 morphants and other ribosomal mutants revealed an activation of the p53 pathway, raising the possibility that a p53 family member could be targeted for DBA treatment (34,37).…”

Functional Links Between Glucocorticoids and Cytokines In DBA

“…In a mouse model of DBA, a high expression of RPS19 can rescue the erythroid development, and the corrected DBA cells show a survival advantage in vivo [11]. As zebrafish hematopoietic regulation is conserved with mammals, zebrafish models have also been reported to be useful in studying DBA [12][13][14], recapitulating many aspects of the DBA phenotype, including hematopoietic specific defects and p53 activation [14]. The list of genes that are mutated in DBA has been updated in 2013, to include ten ribosomal genes and one transcriptional regulator: RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10, RPS26, RPL26, and GATA1 [15].…”