Modeling Diamond Blackfan Anemia in the Zebrafish
Section snippets
Genetics of DBA
RPS19 mutations were first identified in DBA patients over a decade ago.4 The types of mutations seen in patients vary and can include nonsense, missense, frameshift, or splice site mutations. Pathogenic mutations in RPS24,5 RPS17,6 RPL35A,7 RPL5, RPL11, RPS7,8 RPS10, and RPS269 have also been identified in patients. At this point, over half of DBA patients have a known ribosomal protein mutation.3 In addition, mutations in RPL5 and RPL11 are associated with particular congenital abnormalities,8
Mouse and Cell Culture Models of DBA
The first attempt to generate an animal model of DBA was in the mouse (Table 1). With the hypothesis that a mouse heterozygous for the Rps19 mutation would develop DBA-like symptoms, an Rps19 knockout was created. Heterozygous deletion did not have a hematopoietic phenotype, and the homozygous mouse was embryonic lethal.13 Unlike in DBA patients, the wild-type copy of Rps19 is upregulated in the mouse model, compensating for overall RPS19 levels.14 Another mouse model with conditional
DBA Pathogenesis
When ribosomal proteins are mutated or decreased, ribosome biogenesis and rRNA processing are affected.5, 7, 8, 18, 21 Although DBA patients are known to have defects in ribosome biogenesis, the link between these ribosomal protein mutations and DBA symptoms remains unknown. One model suggests that these mutations result in overall translation defects, which might be expected to affect many different tissues. The erythroid lineage may be more susceptible to defects in translational machinery
Developmental Biology
Zebrafish developmental stages have been well-characterized and studied. Embryos are externally fertilized and remain translucent, allowing for easy analysis of phenotypes at very early stages. Gene expression levels can be tested using whole embryo in situ hybridization (ISH), a straightforward assay for tissue-specific gene expression analysis. In addition, embryos develop more quickly than in mammalian models, and primitive red blood cells can be observed in vivo before 48 hours post
Adult Zebrafish Ribosomal Protein Mutants are Predisposed to Cancer
In an effort to identify mutations in genes that are homozygous embryonic lethal, the Hopkins laboratory performed an insertional mutagenesis screen.37 From this screen, 315 mutants were identified, including 28 ribosomal protein mutants.38 Homozygous mutants were initially characterized for developmental defects and led to the identification of novel developmental regulators.39
After characterization of the embryonic phenotypes, the heterozygote adult lines were screened for changes in tumor
Zebrafish Ribosomal Protein Knockdown Models Dba in the Embryo
The homozygous mutants of the insertional mutant library, including the ribosomal protein mutants, were screened for HSC defects at 36–40 hpf by runx1 and c-myb expression.46 Some of the ribosomal protein mutants, including an rps29 mutant, showed decreased HSC marker staining. These data demonstrate that a ribosomal protein mutation can affect HSC formation. The rps29 homozygote also had a defect in arterial specification, assayed by ephrinB2 expression, but the aorta was still present. This
How Zebrafish Models of DBA Can Be Further Developed
One aspect of DBA mutations that has been particularly difficult to model is its apparent haplo insufficiency. A DBA model must reflect the patients' genetics, as patients always maintain one wild-type copy of the ribosomal protein. In the Rps19 knockout mouse, the heterozygote has no phenotype, but the homozygote is embryonic lethal. The zebrafish model has some unique advantages for studying dose effects in vivo. Adjusting the amount of injected morpholino can moderate the level of protein
Summary
Ribosomal protein mutants are found in DBA patients with an erythroid differentiation block. Although there has been significant progress towards understanding how a mutation in a ribosomal protein could cause patient symptoms, many questions remain. The zebrafish model will provide a valuable complement to studies in mammalian systems. Current zebrafish DBA models were critical for identifying a role for p53 in the mutant phenotype. Future work testing candidate pathways and performing
Acknowledgments
The authors would like to thank Katie Kathrein and Charles Kaufman for critical review of the manuscript. L.I.Z. is a founder and stock holder of Fate, Inc. and a scientific advisor for Stemgent.
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Bisphenol-A alters hematopoiesis through EGFR/ERK signaling to induce myeloblastic condition in zebrafish model
2021, Science of the Total EnvironmentCitation Excerpt :The zebrafish (Danio rerio) has proved to be a valuable vertebrate model system in which novel molecular pathways of oncogenesis can be elucidated. Zebrafish has been extensively used for modeling human cancer and hematopoietic disorder, including anemia, thrombocytopenia, bone marrow failure syndromes, leukemia, polycythemia vera and lymphoma (Taylor and Zon, 2011; Seachrist et al., 2016; Kwan and North, 2017; Potts and Bowman, 2017; Gore et al., 2018). On a specific note to hematopoiesis, zebrafish blood contains hematopoietic cells of all myeloid and lymphoid lineages (Willett et al., 1999; Lieschke et al., 2001; Carradice and Lieschke, 2008; Dobson et al., 2008; Renshaw and Trede, 2012).
The transcriptional response of skin to fluorescent light exposure in viviparous (Xiphophorus) and oviparous (Danio, Oryzias) fishes
2018, Comparative Biochemistry and Physiology Part - C: Toxicology and PharmacologyCitation Excerpt :Given these observations, we sought to compare the global transcriptional response in the skin of three commonly utilized biomedical fish models (Xiphophorus maculatus (platyfish), Oryzias latipes (medaka) and Danio rerio (zebrafish)) after exposure to 4100 K “cool white” FL. Zebrafish have been used as neurological disease models (Burgess and Granato, 2007; Galvin, 2006; Montoya et al., 2006; Newman et al., 2007), hematological disease models (Shafizadeh et al., 2002; Taylor and Zon, 2011), tumor models (Liu and Leach, 2011; Avery-Kiejda et al., 2011; Santoriello et al., 2010), and many organ disease models including heart, muscle, kidney and eye (Bassett and Currie, 2003; Bibliowicz et al., 2011; Diep et al., 2011; Gieger et al., 2011; Kawahara et al., 2011; Knöll et al., 2007; Milan et al., 2009; Moosajee et al., 2008; Morris, 2011; Norton et al., 2011; O'Toole et al., 2010; Rihel et al., 2010; Sun et al., 2004; Swanhart et al., 2011). In addition, zebrafish have been used to investigate circadian regulation (Moore and Whitmore, 2014; Noche et al., 2011; Vatine et al., 2011) and light entrainable circadian control of internal organs (Whitmore et al., 1998).
Marrow failure: A window into ribosome biology
2014, BloodCitation Excerpt :Intriguingly, depletion of RPL5 or RPL11 impairs cell-cycle progression in a p53-independent manner.33,34 Mouse21 and zebrafish35 animal models also provide evidence for both p53-dependent and p53-independent pathways in DBA. Indeed, p53 activation appears to be a common downstream stress response pathway activated in response to a variety of different mechanisms underlying nonribosomal marrow failure syndromes such as anemia.36,37
Tbl3 regulates cell cycle length during zebrafish development
2012, Developmental BiologyCitation Excerpt :For example, a mutation in the RNA helicase dead-box 18 (ddx18) results in anemia and neutropenia through p53-mediated G1 arrest and cell death (Payne et al., 2011). In addition, zebrafish models of Diamond-Blackfan anemia have been established demonstrating that loss of specific ribosomal proteins leads to loss of HSPCs and erythrocytes due to upregulation of p53 and cell cycle arrest (Danilova et al., 2008, 2011; Taylor and Zon, 2011; Uechi et al., 2008). These data underscore the importance of cell cycle control during hematopoiesis and suggest that further investigation into specific roles for genes that govern cell cycle speed and progression will be important for full understanding of hematopoietic development.
Clinical Outcomes of Defective Hematopoiesis
2024, Hematopoiesis: Biochemical, Cellular, Molecular, and Genomic PerspectivesAnimal models of Diamond-Blackfan anemia: updates and challenges
2023, Haematologica