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U. Oyarbide et al.
that SDS patients carry at least one hypomorphic SBDS allele.67,72-75 This is consistent with the finding that mice homozygous for null alleles of sbds exhibit early embryon- ic lethality, indicating that SBDS function is an essential for life.30 While conditional knock-outs for sbds have been made, this approach is limited, costly, and time-consuming to generate. Thus, we and others have turned to the zebrafish also to study SDS. Venkatasubramani and Mayer used MO to knockdown sbds in zebrafish embryos, and study the effect in pancreas and myeloid development (Table 4). They observed an alteration in the spatial rela- tionship between endocrine and exocrine pancreas. They also documented abnormal neutrophil distribution in the knockdown zebrafish model.76 In a subsequent study, also using MO, Provost et al. observed that their model fully recapitulated the spectrum of developmental abnormalities observed in SDS patients: loss of neutrophils, skeletal defects, and pancreatic hypoplasia, as well as changes in the ribosomal subunit ratio. In this case, loss of Tp53 did not rescue the developmental defects associated with loss of sbds in zebrafish morphants.77 Our recent work showed that sbds mutants obtained by CRISPR/Cas9 editing phe- nocopied SDS and displayed neutropenia, growth retarda- tion, and atrophy of the pancreas.78
Congenital amegakaryocytic thrombocytopenia
Congenital amegakaryocytic thrombocytopenia is a rare autosomal recessive condition characterized by thrombo- cytopenia, absence of megakaryocytes, and occasional evolution to aplastic anemia or leukemia.79,80 Mutations in MPL have been described as the cause of CAMT.81 MPL gene encodes for myeloproliferative leukemia protein (CD110), the receptor for thrombopoietin. Mice with genetic ablation of Mpl showed normal development but a deficiency in megakaryocytes and severe thrombocy- topenia.82 In zebrafish, disruption of mpl caused a severe reduction in thrombocytes (platelet equivalents), bleeding, and a decrease in HSCs. By phenocopying the human dis- ease, affected zebrafish provide an accurate model to study this disease and for drug screening.83 Reduction in HSCs and repopulation defects in affected zebrafish demonstrate that c-Mpl function in hematopoiesis is high- ly conserved. Moreover, the partial rescue of thrombocyte number by IL-11 provides a model to finely dissect JAK/STAT signaling in thrombopoiesis.
Severe congenital neutropenia
Severe congenital neutropenia is a group of heteroge- neous genetic disorders characterized by a maturation arrest at the promyelocyte stage of granulopoiesis and a high propensity to develop MDS/AML.84 Over the past eighteen years, the following mutations have been identi-
fied as causing SCN: ELANE, GFI1, HAX1, VPS45, JAGN, CSF3R, and WAS. ELANE is the most commonly mutated gene in SCN, but there is no zebrafish ortholog. However, zebrafish has proven to be a powerful model to validate and characterize the function of newly described gene candidates for SCN. Vacuolar Protein Sorting 45 Homolog (VPS45) encodes a protein associated with protein traf- ficking into distinct organelles. Biallelic mutations in this gene are the cause of SCN5. A zebrafish model of vps45 knockdown also showed a large decrease in neutrophils.85 Mutations in CSF3R cause SCN7.86 Pazhakh et al. mutated csf3r in zebrafish to study the effect on neutrophil produc- tion. They found that csf3r zebrafish mutants survive until adulthood with a 50% reduction in neutrophils and a sub- stantial reduction in myeloid cells in the kidney marrow.87 Recently, SRP54 mutations have been identified as the sec- ond most common cause of SCN (with some features of SDS).88,89 Knockdown of SRP54 in zebrafish recapitulated the human phenotype of neutropenia, chemotaxis defect, and pancreatic exocrine insufficiency.88
Conclusions
Despite the identification of specific gene mutations and pathway involvement for the great majority of patients with IBMFS, little is known about how they result in sin- gle or multiple lineage cytopenias. Furthermore, very little is known about co-operating mutations that effect trans- formation to MDS, AML, or solid tumors. Patient-based studies are problematic owing to the rarity of these disor- ders and to the long latency before bone marrow failure or malignancy. Zebrafish provide a relatively inexpensive, rapidly developing, vertebrate model organism. Despite some differences in their respective hematopoietic organs, mutations or silencing of relevant zebrafish genes pheno- copies human IBMFS. Studies on gene mutations or sup- pression in zebrafish have validated the role of ribosome biogenesis, and advanced the hypothesis that the TP53 pathway plays a major role in the pathophysiology of some of the IBMFS. Zebrafish modeling may also con- tribute to drug development, as suggested by studies on L- leucine and SMER28 for DBA.
Acknowledgments
SJC is supported by funding from NIH R01 HL128173, NIH R21 CA159203, Department of Defense Bone Marrow Failure Idea Development Award BM140102, Shwachman-Diamond Syndrome Foundation, Connor’s Heroes, and the CURE Childhood Cancer Foundation. Due to space restrictions, the authors deeply regret not being able to cite all of our colleagues’ publications.
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