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U. Oyarbide et al.
involved in bone marrow failure. One p53-independent effect may be translational dysfunction. Zebrafish can provide a model organism to identify Tp53-independent pathways that contribute to marrow failure mice or humans.
Zebrafish may also be a valuable model organism for drug development for DBA treatment. Several groups have tested the hypothesis that L-leucine and L-arginine can stimulate translation via the mTOR pathway and res- cue affected DBA fish. Treatment of rpl19 and rpl14 zebrafish morphants with L-leucine improved develop- mental defects and hemoglobin levels.45 Yadav et al. res- cued the morphological defects of Rpl35a-deficient embryos and were able to improve erythroid cell number.42 They concluded that translation deficit, not Tp53 activation, is the primary defect perturbing erythro- poiesis.42 While there have been anecdotal reports of leucine stimulation of erythropoiesis in DBA patients,46 definitive clinical trial results are still pending. Another study found that RAP-011, an activin receptor ligand trap, partially restored erythropoiesis in rpl11 morphants as well as rpl11 and rpl19 mutants.47 Zebrafish also provided an in vivo model for further drug development of SMER28, a small molecule inducer of ATG5-dependent autophagy.48 Given these results, we await clinical trans- lation of SMER28 as a potential treatment for DBA.
Dyskeratosis congenita
Dyskeratosis congenita is associated with abnormal skin pigmentation, nail dystrophy, and oral leukoplakia. DC patients may have other organ involvement, including the pulmonary, gastrointestinal, skeletal, neurological, immunological, and ophthalmological systems. Eighty- five percent of DC patients experience bone marrow fail- ure, which accounts for much of the DC-related mortality. Other causes of mortality include infections, pulmonary complications, and hematologic and non-hematologic malignancies.49-51
Dyskeratosis congenita is a genetically heterogeneous disorder, showing autosomal recessive, autosomal domi- nant, and X-linked inheritance. So far, at least 21 mutated genes have been identified that can cause DC: DKC1, TERC, TERT, NHP2, NOP10, CTC1, WRD79, TR, NOLA2, NOLA3, PARN, TPP1, POT1, CTC1, USB1, TCAB1, RTEL1, ACD, PARN, WRAP53 and TINF2 (http://telomerase.asu.edu/diseases.html).51-53 The X-linked DKC1 has a more severe phenotype compared with the autosomal dominant forms. Although there is a broad consensus that DC results from stem cell renewal failure due to defective telomere maintenance, some mutated genes (e.g. TERT, TERC, and DKC1) are required for pre- rRNA processing.2,49,50,54 How telomerase activity and impaired ribosomal biogenesis contribute to the patho- physiology of DC is still not known. Telomeres are com- plex DNA-protein structures at the end of chromosomes, and they shorten with each cell division. When telom- eres become critically short, a DNA damage response is activated, causing cell cycle arrest or death. In humans, telomerase-based telomere elongation is the major mechanism that counteracts this process of continuous telomere shortening. In peripheral white blood cells, rapid telomere shortening occurs within the first year of life, followed by a more gradual decline over time.49 Genetic diseases that cause telomerase deficiency are associated with premature aging and cancer susceptibili-
ty. As in humans, zebrafish chromosomes possess telom- eres that progressively decline with age, reaching lengths in old age comparable to those observed when telom- erase is mutated.55 Several studies have helped to charac- terize its well-conserved molecular and cellular physiolo- gy. Different zebrafish mutants and morphants for telomere and telomerase research showed shorter lifes- pan, shorter telomeres, and different affected tissues (mainly brain, blood, gut and testes). These results make zebrafish an excellent model to unravel the connection between telomere shortening, tissue regeneration, aging and disease.55,56
Amsterdam et al. isolated the nop10hi2578 mutant allele where a viral insertion within the first intron resulted in nop10 decreased expression. This mutation is homozy- gously lethal by 5 dpf.21 nop10 encodes for a protein involved in 18S rRNA processing and is also part of the telomerase complex. Pereboom et al. observed that nop10 loss in this mutant line resulted in a failure of the 18S rRNA to be properly processed, which led to the instabil- ity of the 40S ribosomal subunit. Due to the loss of 18S RNA, ribosomal proteins cannot be incorporated into a ribosome subunit and interact with other proteins, includ- ing the E3 ubiquitin ligase Mdm2. Mdm2 regulates Tp53 by promoting its ubiquitination and degradation. By bind- ing to Mdm2, Rps7 enhances the E3 ubiquitin ligase activ- ity of Mdm2 that promotes the degradation of Rps7. Furthermore, they observed that an increase in Tp53-spe- cific apoptosis is coupled to the increased binding of Mdm2 to the Rps7. They observed that nop10 mutants failed to form HSCs, a phenotype that is rescued by intro- ducing a loss-of-function tp53 mutation. However, they detected no changes in telomere length in nop10 mutants.53 They concluded that the cytopenia(s) of DC could be the result of ribosome biogenesis defects. This would lead to Tp53-mediated apoptosis of HSCs during early develop- ment, caused partially by the association of Rps7 with Mdm2.53
Two different approaches were used by Zhang et al. to study DC in zebrafish. First, MO-mediated knockdown was used to study the mechanisms whereby dkc1 mor- phants result in HSC failure. Second, they performed retroviral-insertional mutagenesis of nola1. NOLA1 encodes for GAR1, involved in rRNA maturation, and is also a key component telomerase complex. No mutations in NOLA1 have been described in DC patients so far, but suspicion should be aroused in individuals with unex- plained marrow failure or fibrosis. Both zebrafish models resulted in reduced hematopoiesis with reduction in runx1 and c-myb, increased tp53 expression, and defective ribo- somal biogenesis without detectable changes in telom- erase function. Their findings suggest that a telomerase- independent, Tp53-dependent mechanism contribute to hematopoietic failure in DC.50
Henriques et al. and Anchelin et al. studied the zebrafish telomerase reverse transcriptase tert mutant. These mutants develop normally for the first six months, but progressively develop tissue degeneration (gastrointestinal atrophy, loss of body mass, inflammation, a decrease in total blood cells and cell proliferation), and die premature- ly. They also observed a Tp53-dependent response with increased transcripts of puma, cdkn1a, and ccng1a. Upregulation of cell cycle arrest inhibitors led to a G1 arrest and senescence. To study the effect of Tp53 in tert mutants, they created a double mutant tert-/-, tp53-/- and
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haematologica | 2019; 104(1)