Session: 201. Granulocytes, Monocytes, and Macrophages: Poster I
Hematology Disease Topics & Pathways:
Bone Marrow Failure Syndromes, Inherited Marrow Failure Syndromes, hematopoiesis, Diseases, Biological Processes
Methods. We used CRISPR/Cas9 editing to generate a mutation in exon 7 of efl1 zebrafish, resulting in a premature termination codon that disrupts the GTP-binding domain. We bred efl1+/- fish and collected larvae at 5 and 15 days post fertilization (dpf).
Results. Genotype frequency of eggs fertilized from heterozygous carriers followed the expected Mendelian distribution. However, efl1-/- survival declined starting from 15 dpf. Standard length in efl1-/- was significantly reduced at 15 dpf, but not at 5 dpf. Neutrophils were significantly decreased in efl1-/- fish in comparison with those in efl1+/+ and efl1+/- siblings. The median number of neutrophils at 5 dpf was 36 (range, 17–79) in efl1-/-, whereas the median in efl1+/+ and efl1+/- was 93 (range, 29–193) and 84 (range, 17–151), respectively (P < .0001). At 5 dpf, RT-qPCR using whole larvae RNA showed significantly increased (4-8 fold) cdkn1a mRNA in efl1-/- fish compared to efl1+/+ and efl1+/- siblings. There was no significant upregulation of tp53 mRNA in efl1-/-. Accumulation of Eif6 protein in efl1-/- fish was found at 15 dpf, although eif6 mRNA were not significantly changed among efl1 genotypes. Because we reported in our sbds-null zebrafish aberrant fatty acid and cholesterol regulatory gene expression, we examined their expression in this efl1-null zebrafish. By 15 dpf, srebp1 mRNA was significantly higher in efl1-/-, and pparg and fasn also tended to be higher in efl1-/- in comparison with efl1+/+ and efl1+/-.
Conclusions. Zebrafish lacking efl1 phenocopied some of the molecular and morphologic features of SDS. Additionally, results from efl1-/- zebrafish were consistent with those from sbds-/- zebrafish strains, emphasizing a common molecular pathway induced by the dyad of EIF6 dissociating factors. Ongoing studies, which will be presented, are evaluating skeletal abnormalities and pancreatic atrophy in the efl1-/- zebrafish. Interestingly, sbds-null and efl1-null fish survived longer than eif6-null, suggesting more critical functions of EIF6. Altogether our data and presented elsewhere advance the hypothesis that CDKN1A and EIF6 contribute to the pathophysiology of SDS due to either mutations in SBDS or EFL1.
Disclosures: No relevant conflicts of interest to declare.
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