Molecular Mechanism Research Of Hematopoietic Failure Due To RPL5and RPL11Deficiency In A Zebrafish Model Of Diamond-blackfan Anemia

Diamond-Blackfan anemia is a rare inherited bone marrow failure syndrome that ischaracterized by pure red-cell aplasia and associated physical deformities. The syndromewill develop soon after the patients’ birth and threaten their health. Previous studies havefound that more than50%of the patients presented with ribosomal gene mutations, andidentified a variety of pathogenic mutant ribosomes genes. About20%of the patientsscreened had mutations in RPL5or RPL11. However, the mechanisms by which thedefects of RPL5and RPL11regulate hematopoiesis in DBA have not been fullyelucidated.In this study, we used the zebrafish as a model organism and respectively obtainedthe RPL5-deficient and RPL11-deficient zebrafish embryos through micro-injection ofRPL5MO (morpholino) and RPL11MO. Then we compared the whole genome RNA-Seqdata of the RPL5-deficient and RPL11-deficient zebrafish embryos with the controls,tested the hemoglobin and granulocytes synthesis with o-dianisidine staining and sudanblack staining orderly, detected the expression level of different blood cells and vesselrelated marker gene by whole mount in situ hybridization and RT-PCR, and then detectedthe expression level of pAKT (phosphorylated AKT) and pERK (phosphorylated ERK)through western blotting. Finally we coinjected RPL11MO and p53MO, RPL11MO andp53MO, or their respective control MOs in turn, acquiring the data through experimentalmethods above.We found the RPL5-and RPL11-deficient zebrafish embryos result in hematopoieticand developmental abnormalities that resemble DBA, such as haematogenesis disorder,morphological mild malformation, and then we observed the expression level ofhematopoietic stem cells marker genes decreased significantly, such as c-myb and runx1,etc. Moreover, we observed that coinjection of RPL11MO and p53MO, or RPL11MOand p53MO could rescue the haematogenesis defects partially, and reduce the pAKT andpERK level slightly.From this study, we can conclude that DBA is a systemic and complex disease due toribosomal protein defects. RPL5and RPL11deficiencies link to DBA closely, which cause HSC and hematopoiesis dysfunction consequentially. We also confirmed that the p53pathway involves in RPL5and RPL11deficiencies which lead to a lack of hematopoiesis,and RPL5and RPL11deficiencies may give rise to HSC malfunction by influencing thePI3K/AKT and MAPK/ERK signaling pathway. Our work has laid foundation in thezebrafish DBA model, clarifying the pathogenesis of DBA detailly and it is important todetect the potential pathogenic genes and provide a new way of the treatment for humanDBA.