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IBMFS in zebrafish
viral insertion,20,21 gene function can be studied by trans- genic expression or genome editing by transcription acti- vator-like effector nucleases (TALEN) or Cas nucleases act- ing on clustered, regularly interspaced, short palindromic repeats (CRISPR). Gene expression can be silenced tem- porarily and early during development by injection of morpholino antisense nucleotides (MO).
Zebrafish have provided a useful model organism for a quick validation and study of human disease candidate genes, including those involved in the pathophysiology of IBMFS (Table 4). MO-mediated knockdown was widely used to probe gene function, though this method has lim- itations. Phenotype of morphants (MO-injected animals) can differ and is often more severe than those of the cor- responding mutants. There could be different reasons for this: 1) phenotypic rescue of zygotic mutants by maternal wild-type mRNA; 2) off-target effects of the MO; 3) hypo- morphic nature of the mutant allele analyzed; or 4) genetic compensation in mutants but not in morphants (see Stainier et al.22). Moreover, injection of MO can cause Tp53 activation and cell death.23 In some instances, cell death can be prevented by simultaneous blocking of p53 by a second MO. This may lead to a misinterpretation of
results, particularly in processes that depend on the Tp53 DNA damage response pathway (reviewed below). In some cases, results of MO knockdowns were not recapit- ulated with the genome editing techniques.24 Close exam- ination of the differences in gene expression revealed a novel compensation mechanism that operates only after mutation but not after MO knockdown (Table 5).25
Diamond-Blackfan anemia
Diamond-Blackfan anemia is characterized by red cell hypoplasia, erythroid macrocytosis, and markedly reduced erythroid precursors in the bone marrow. Other hematopoietic lineages are usually normal at birth,26 but they may be affected later in childhood/adolescence.27 In
Table 2. Gene and protein nomenclature among species
Human
Mouse
Zebrafish
Gene symbol
sbds
sbds
sbds
Protein symbol
SBDS
SBDS
Sbds
Figure 1. Comparison of developmental hematopoiesis in humans and zebrafish. Primitive and definitive hematopoiesis occurs in both species. In human, hematopoietic stem cells (HSC) originate in aorta-gonad-mesonephros (AGM) and placenta, from where they colonize fetal liver and finally the bone marrow. In zebrafish, primitive hematopoiesis starts after hemangioblast formation around 12 hpf in the anterior lateral mesoderm (ALM) and posterior lateral mesoderm (PLM). Later, HSCs originate in the AGM and then mobilize to caudal hematopoietic tissue (CHT) prior to their final destination of the kidney (modified from Teittinen et al.12 and de Jong and Zon14).
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