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IBMFS in zebrafish
nucleosides resulted in downregulation of tp53 and its tar- gets with normalization of ada and xdh levels. Interestingly, DBA patients show increased erythrocyte adenosine deaminase activity.38
Zhang et al. generated two zebrafish mutants using TALENs for rps19 and rps11. The knockout of both rps19 and rps11 resulted in the erythroid defects similar to DBA, such as lack of mature red blood cells (RBCs) and Tp53 activation. The mutants had significantly reduced produc- tion of globin proteins accompanied by either increased or unaffected level of mRNA transcripts. Furthermore, they observed decreased HSCs at 3 dpf in rps19 mutants and hemoglobin levels by 4 dpf. The authors concluded that this reduction in RBCs may be caused by a decreased cell survival and/or production of definitive HSCs.30 Similarly, Rowel et al. created a 5 bp deletion in rps19 zebrafish mutant using TALENs. Homozygous rps19 mutants showed developmental anomalies and anemia, and were dead by 5 days post fertilization (dpf). However, rps19 heterozygotes showed no difference to their wild-type siblings. Interestingly, exposure to cold stress during the first dpf resulted in a reduced number of RBCs.
To further investigate the biological functions of RPS7, Duan et al. used MO to knockdown rps7 in zebrafish.39 In rps7-deficient embryos, mdm2 and tp53 were activated, inducing the expression of downstream target genes involved in p53 pathway (bik, bax and puma, cdkn1a, and ccng1). rps7 morphants showed severe anemia with reduced expression of gata1 and the mature erythroid marker αe3 at 24 hours post fertilization (hpf). A marked suppression of hemoglobin at 48 hpf was observed, indi- cating that the deficiency of Rps7 might cause abnormal proliferation and/or differentiation of erythroid progeni- tors. There were also severe defects (short body length, tissue necrosis, and curved tail). Furthermore, simultane- ous knockdown of the tp53 by co-injecting a tp53 MO
Table 5. Comparison between morphants and mutants.22-24 Morphant
resulted in partial rescue of morphological abnormalities. The lower levels of gata1 and α-E1 globin were partially rescued in the co-injected embryos, even though tp53, cdk1a, and mdm2 were still up-regulated.39
The contribution of tp53 to the pathological develop- ment of bone marrow failure syndromes may be tissue- and mutation-specific. Antunes et al. studied the effect of different rps7 and rpl11 mutations in zebrafish. rps7 mutant showed a stronger phenotype due to less mater- nal contribution of rps7 comparing to rpl11 mutant. Both mutants had severe anemia, morphological abnormali- ties, and increased apoptosis. Injection of p53 MOs res- cued the apoptosis and the morphological phenotypes; however, it was unable to rescue anemia.40 Taylor et al. showed that rps29 mutants had defects in RBC develop- ment and increased apoptosis. Mutant embryos showed upregulation of tp53 and cdk1a expression. Mutation of tp53 in homozygous rps29 mutant embryos reversed the apoptotic and hematologic phenotypes. However, mutat- ed tp53 did not fully rescue the embryonic lethality of rps29 mutants, suggesting that tp53-independent mecha- nisms were affected by rps29 knockdown.41 Yadav et al. knocked down five ribosomal protein genes (two DBA- associated, rpl35a and rps24, and three non-DBA-associat- ed, rps3, rpl35 and rplp1), and analyzed these deficiencies on morphology and erythrocyte number in the presence and absence of p53 using MOs. They showed that any ribosomal protein deficiency led to anemia in zebrafish. Elimination of Tp53 function did not significantly affect the anemia, despite improving non-hematopoetic pheno- types.42 DBA zebrafish models have helped identify MDM2-ribosomal protein interactions, which may inter- fere with MDM2 inhibition to p53 function. p53 rescue of severe anemia in ribosomal protein deficiency zebrafish models varies (Table 6).31,35,43,44 Altogether, these findings suggest that there are p53-independent mechanisms
Mutant
Permanent changes in DNA
Genomic DNA
Less severe maternal mRNA 6-8 months
Less off target effects Yes Non-affected
Effect
Affects
Phenotype
Time to create
Side effects
Genetic compensation p53 pathway
Morphants versus mutants Knock down
RNA transcripts
More severe maternal mRNA block by MO 1-3 days
More off-target effects
No
Affected
Table 6. RP deficiency and p53 rescue in zebrafish models.
RP
rps19 morphant
rpl11 morphant
rps7 morphant
Rps7 mutant
Rps29 mutant
rps24 & rpl35a morphants
Severe anemia
Yes
N/A Yes Yes Yes Yes
Developmental malformations
Yes
Yes Yes Yes Yes Yes
Type of p53 rescue
p53 rescue of anemia
p53 rescue of Ref other phenotypes
p53MO No Yes
p53MO N/A Yes p53 MO Partial Partial p53MO No Yes
p53M214K Yes Yes p53M214K No Yes
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35 40 36 93 42
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