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      Atm deficient zebrafish model reveals conservation of the tumour suppressor function and a role in fertility

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          Abstract

          Biallelic loss-of-function variants in ATM (Ataxia Telangiectasia Mutated) cause Ataxia Telangiectasia (AT), a rare disorder associated with cerebellar degeneration and ataxia, cancer predisposition, infertility, growth retardation, etc. ATM is a phosphoinositide 3-kinase-related kinase (PIKK) with a role in DNA repair and maintenance of genome stability. Studying a multisystem genetic disease like AT requires animal models to ascertain its pathogenesis at the level of tissues, organs and the organism. Due to its small size, cheap maintenance, large progeny, rapid development and initial transparency, zebrafish (Danio rerio) is an increasingly popular vertebrate model organism, suitable for genetic modifications and large-scale in vivo therapeutic screens as embryos are chemically permeable to small compounds. Currently, no zebrafish model for AT exists. 1 We generated atm knock-outs through CRIPSR-Cas9 mutagenesis. We show that atm conserved its function as a tumour suppressor gene and is involved in gametogenesis and fertility. Therefore, this mutant is of great value for further studies investigating the role of atm in reproduction and tumorigenesis. Zebrafish atm evolved to a single copy after the teleost genome duplication, which appears distinctive for DNA damage repair genes. 2 Human ATM and zebrafish atm both comprise 62 coding exons with high amino acid conservation in functional domains (Fig. 1A and Table S1). Therefore, ATM functions may be conserved in zebrafish and atm deficiency might recapitulate human disease. Figure 1 Characteristics of Atm deficient zebrafish. (A) Zebrafish and human Atm/ATM structure. atm mutations introduced by CRISPR-Cas9 mutagenesis are indicated. Protein domains of human ATM (3056 aa) and zebrafish Atm (3091 aa) are highly conserved. The encoded proteins display an overall AA sequence identity of 54% with a higher conservation for the important functional protein domains (yellow = TAN (Tel1/ATM N-terminal motif: LxxxKxxE/DRxxxL); black stripe = c-ABL; orange = FAT (FRAP-ATM-TRRAP) domain; blue = PI3K (phosphoinositide 3-kinase-like) domain; green = FATC (FAT C-terminal). These domains display a homology of 77, 78, 60, 82 and 94% respectively, with also Ser1981, an important autophosphorylated site, being preserved at AA position 2012 in zebrafish (B) atm deficient zebrafish developed exclusively as males (P < 10−5, Fisher's exact test). Mendelian inheritance laws rule out embryonic lethality. Black line = 1 cm. (C) Histology of zebrafish gonads. Zebrafish atm wild types and heterozygotes have fully functional gonads. Oocytes in all stages of maturation are present, while testes have adult sperm in their lumen. Zebrafish atm mutants do not develop ovaria. Zebrafish atm testis are devoid of spermatids and spermatozoa. Red L = lumen, I = Stage I oocytes, II = Stage II oocytes, III = Stage III oocytes. (D) Wild-type testis has spermatogonia, spermatocytes, spermatids and spermatozoa. In contrast, atm-mutants lack spermatids and spermatozoa in their lumen. Spermatocytes are arrested in the bouquet phase of meiosis, and some spermatocytes appear pyknotic. sg: spermatogonia, sc: spermatocytes, st: spermatids, sz: spermatozoa, pk: pyknosis. (E) Caspase-3 immunostaining of testis clearly shows cleaved caspase-3 positive apoptotic cells in atm-mutants. Black arrows indicate cells undergoing apoptosis. atm cmg33/cmg34 ;tp53 zdf1/zdf1 mutant zebrafish are prone to develop tumours. (F) Age of tumour onset is significantly lower in atm cmg33/cmg34 ;tp53 zdf1/zdf1 zebrafish compared to atm +/- ;tp53 zdf1/zdf1 and atm +/+ ;tp53 zdf1/zdf1 zebrafish. ∗: P < 10−3, ∗∗: P < 10−4. Unpaired t-test was used. Error bars indicate 95% Confidence Interval (CI). In GI-III fish with protruding tumours are shown (inset: tumour location indicated with red arrow; main figure: showing histopathological image of tumour (G.I) Malignant peripheral nerve sheath tumour (MPNST) in the eye of a double mutant zebrafish. The tail of this zebrafish was deformed since early development (G.II) undifferentiated spindle cell sarcoma (US) growing in the tail (G.III) MPNST in the coelom invading the intestinal lining. Fig. 1 Through CRISPR/Cas9 mutagenesis we generated two heterozygous atm knock-out mutant zebrafish lines, with premature stop codons in exon 4 (atm cmg34) and 9 (atm cmg33) (Fig. 1A; Fig. S1 and Table S2). For all experiments, these lines were in-crossed to obtain compound heterozygous mutants (atm cmg33/cmg34, “atm-mutants”), avoiding potential homozygosity of CRISPR-induced off-target mutations. Atm deficiency is not embryonically lethal: offspring of atm cmg33/+ xatm cmg34/+ (n = 77) displayed 27% wild types, 51% heterozygotes and 22% mutants (Fig. 1B), in agreement with Mendelian inheritance (Chi 2  = 0,80). Equal rates of males:females were found for wild types and heterozygotes; however, atm cmg33/cmg34 fish developed exclusively as males. This all-male phenotype was found over multiple crosses and generations of atm-mutants. Atm-mutant males were able to induce egg laying in wild-type females, but eggs remained unfertilized (Fig. S2). H&E (hematoxylin and eosin) stained histological sections of gonads displayed male testes in all atm-mutants (Fig. 1C) but lacked spermatids and adult spermatozoa (Fig. 1D), while wild-type testes contained cells at all stages of differentiation during spermatogenesis. Additionally, testes of mutants displayed large groups of spermatocytes in the bouquet phase of meiosis and some spermatocytes appeared pyknotic, suggesting apoptosis. Apoptosis was confirmed by Caspase-3 immunostaining and TUNEL labelling (Fig. 1E; Fig. S3) and indicates that zebrafish atm is essential in spermatogenesis. Zebrafish lack identifiable heteromorphic sex chromosomes. The current model hypothesizes that zebrafish sex is partially determined by the number of primordial germ cells (PGC), with reduced numbers causing zebrafish to become males. The Fanconi anaemia (FA) pathway is required for PGC to survive and thus allow female sex to occur. 2 ATM is attributed multiple roles in homologous recombination (HR), essential for repair of crosslinks through the FA pathway. However, ATM is also implicated in control of meiotic double strand break (DSB) formation. Absence of functional ATM could lead to over-activation of SPO11-mediated DSBs, ultimately leading to apoptosis. 3 The all-male phenotype suggests that PGC of juvenile zebrafish require atm for survival. Next, we investigated if apoptosis of PGC causes the all-male phenotype in atm-mutants by disabling p53-mediated apoptosis. Hereto we generated a double knock-out atm cmg33/cmg34 ;tp53 zdf1/zdf1 model. tp53 zdf1/zdf1 mutants displayed equal rates of males:females, while atm-mutants with a functional tp53 allele developed exclusively as males (Table S3). However, atm cmg33/cmg34 ;tp53 zdf1/zdf1 males could not fertilize eggs of wild-type females (Fig. S4A). Histology confirmed that spermatids and spermatozoa were absent in these zebrafish, similar to atm cmg33/cmg34 ; tp53 +/+ zebrafish (Fig. S4B). atm cmg33/cmg34 ;tp53 zdf1/zdf1 females produced some fertilized eggs, but oocytes likely acquired genomic aberrations, as all embryos died within 1 day post fertilization (Fig. S4C). Histology of the ovaries appeared normal (Fig. S4D). As atm +/+ ;tp53 zdf1/zdf1 and atm +/- ;tp53 zdf1/zdf1 females were able to produce healthy offspring, we conclude that atm is essential for the viability of zebrafish oocytes. Therefore, atm plays a role in zebrafish sex development and fertility. Infertility has been reported in AT individuals and multiple Atm-deficient animal models. Another key characteristic of AT patients is their increased cancer risk (30%–40% lifetime risk). We sectioned 12 atm-mutants of 16 months old but could not find tumours/neoplasia. This is not surprising, since knocking out tumour suppressors in zebrafish does not cause the same increased cancer risk as in humans: TP53 and BRCA2 germline mutations confer a 70%–100% cancer risk in humans. In zebrafish, tp53 deficiency caused a 40% cancer risk, 4 and only 30% of brca2-deficient zebrafish displayed testicular neoplasia at 16 months. 5 To accelerate tumour formation, we investigated atm-deficiency in a tp53-mutated background. atm cmg33/cmg34 ;tp53 zdf1/zdf1 zebrafish developed tumours much faster (average: 9.0 months) compared to atm +/- ;tp53 zdf1/zdf1 (average: 14,1 months, p < 10−4) or atm +/+ ;tp53 zdf1/zdf1 zebrafish (average: 13,4 months, p < 1 0−3) (Fig. 1F and Table S4), suggesting that atm conserved its tumour suppressor role and that atm deficiency accelerated tumorigenesis in tp53-mutant zebrafish. Malignant peripheral nerve sheath tumours (MPNST), undifferentiated spindle cell sarcomas (US) and seminomas were found (Fig. 1G), which are standard for tp53 zdf1 . AT patients regularly develop leukaemia, which we did not observe in atm-mutants. Similarly, brca2 −/− zebrafish do not present with haematological tumours while Fanconi anaemia patients develop such malignancies. Studying leukaemia in atm-mutants may require transgenic lines containing oncogenic drivers or another type of tp53 variant: tp53 zdf1 contains a missense variant and MPNSTs are typically found, while tp53 del/del harbours a deletion and showed a high proportion of leukaemia. 4 The ataxia-phenotype of AT patients was not reproduced in zebrafish atm-mutants, based on swim movements in a rotating chamber at the age of 6 or 12 months (Fig. S5A). This is in contrast to some murine, rat and porcine models. We cannot rule out that degenerative phenotypes only manifest at later ages. Alternatively, atm-deficient zebrafish may display a subtle phenotype, requiring more sophisticated methods, like studying the optokinetic response, as AT patients have trouble reading due to impaired coordination of the eye movement. The cerebellum of AT patients displays neurodegeneration, hallmarked by loss of Purkinje cells. Histological analysis of the cerebellum in aged atm-mutants revealed a normal Purkinje cell count and molecular layer thickness (Fig. S6A). Also in murine models, histology was unable to show abnormal cerebellar architecture. A higher complexity of the brain may be needed for atm to play a major role in neurodegeneration. To come to definite conclusions additional tests with larger sample sizes and zebrafish of different ages may be required. In contrast to human AT patients, growth retardation was not displayed in adult (12 months old) atm-deficient zebrafish compared to age-matched wild-type control males (Fig. S7). Generally, zebrafish with loss of DNA damage response (DDR) genes lack this phenotype. Zebrafish may suffer less from dysphagia, which in children leads to reduced caloric intake and compromises growth. Other explanations could be a role of modifier genes, or a lack of environmental challenges in laboratory settings. We cannot fully exclude residual functionality of the mutant protein as suitable Atm antibodies to check for residual protein were not available. In conclusion, despite the absence of a clear neurodegenerative phenotype, we show that zebrafish atm is required for sex-determination and fertility and acts as a tumour suppressor gene. The model could be useful to discern the role of this kinase in genome stability maintenance and tumour suppression and may be used to discover novel compounds targeting ATM deficiency. Conflict of interests Authors declare no conflict of interests. Funding This work was supported by ‘Kom op Tegen Kanker – Emmanuel Van der Schueren’ (No. 365M02318) and by ‘UGent- Bijzonder OnderzoeksFonds’ (No. BOF15 GOA/011).

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          Most cited references5

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          ATM and PRDM9 regulate SPO11-bound recombination intermediates during meiosis

          Meiotic recombination is initiated by SPO11-induced double-strand breaks (DSBs). In most mammals, the methyltransferase PRDM9 guides SPO11 targeting, and the ATM kinase controls meiotic DSB numbers. Following MRE11 nuclease removal of SPO11, the DSB is resected and loaded with DMC1 filaments for homolog invasion. Here, we demonstrate the direct detection of meiotic DSBs and resection using END-seq on mouse spermatocytes with low sample input. We find that DMC1 limits both minimum and maximum resection lengths, whereas 53BP1, BRCA1 and EXO1 play surprisingly minimal roles. Through enzymatic modifications to END-seq, we identify a SPO11-bound meiotic recombination intermediate (SPO11-RI) present at all hotspots. We propose that SPO11-RI forms because chromatin-bound PRDM9 asymmetrically blocks MRE11 from releasing SPO11. In Atm –/– spermatocytes, trapped SPO11 cleavage complexes accumulate due to defective MRE11 initiation of resection. Thus, in addition to governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing.
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            brca2 in zebrafish ovarian development, spermatogenesis, and tumorigenesis.

            Humans with inherited mutations in BRCA2 are at increased risk for developing breast and ovarian cancer; however, the relationship between BRCA2 mutation and these cancers is not understood. Studies of Brca2 mutation by gene targeting in mice are limited, given that homozygous Brca2 mutation typically leads to early embryonic lethality. We established a zebrafish line with a nonsense mutation in brca2 exon 11 (brca2(Q658X)), a mutation similar in location and type to BRCA2 mutations found in humans with hereditary breast and ovarian cancer. brca2(Q658X) homozygous zebrafish are viable and survive to adulthood; however, juvenile homozygotes fail to develop ovaries during sexual differentiation. Instead, brca2(Q658X) homozygotes develop as infertile males with meiotic arrest in spermatocytes. Germ cell migration to the embryonic gonadal ridge is unimpaired in brca2(Q658X) homozygotes; thus, failure of ovarian development is not due to defects in early establishment of the embryonic gonad. Homozygous tp53 mutation rescues ovarian development in brca2(Q658X) homozygous zebrafish, reflecting the importance of germ cell apoptosis in gonad morphogenesis. Adult brca2(Q658X) homozygous zebrafish are predisposed to testicular neoplasias. In addition, tumorigenesis in multiple tissues is significantly accelerated in combination with homozygous tp53 mutation in both brca2(Q658X) homozygous and brca2(Q658X) heterozygous zebrafish. These studies reveal critical roles for brca2 in ovarian development and tumorigenesis in reproductive tissues.
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              Multiplexed CRISPR/Cas9-mediated knockout of 19 Fanconi anemia pathway genes in zebrafish revealed their roles in growth, sexual development and fertility

              Fanconi Anemia (FA) is a genomic instability syndrome resulting in aplastic anemia, developmental abnormalities, and predisposition to hematological and other solid organ malignancies. Mutations in genes that encode proteins of the FA pathway fail to orchestrate the repair of DNA damage caused by DNA interstrand crosslinks. Zebrafish harbor homologs for nearly all known FA genes. We used multiplexed CRISPR/Cas9-mediated mutagenesis to generate loss-of-function mutants for 17 FA genes: fanca, fancb, fancc, fancd1/brca2, fancd2, fance, fancf, fancg, fanci, fancj/brip1, fancl, fancm, fancn/palb2, fanco/rad51c, fancp/slx4, fancq/ercc4, fanct/ube2t, and two genes encoding FA-associated proteins: faap100 and faap24. We selected two indel mutations predicted to cause premature truncations for all but two of the genes, and a total of 36 mutant lines were generated for 19 genes. Generating two independent mutant lines for each gene was important to validate their phenotypic consequences. RT-PCR from homozygous mutant fish confirmed the presence of transcripts with indels in all genes. Interestingly, 4 of the indel mutations led to aberrant splicing, which may produce a different protein than predicted from the genomic sequence. Analysis of RNA is thus critical in proper evaluation of the consequences of the mutations introduced in zebrafish genome. We used fluorescent reporter assay, and western blots to confirm loss-of-function for several mutants. Additionally, we developed a DEB treatment assay by evaluating morphological changes in embryos and confirmed that homozygous mutants from all the FA genes that could be tested (11/17), displayed hypersensitivity and thus were indeed null alleles. Our multiplexing strategy helped us to evaluate 11 multiple gene knockout combinations without additional breeding. Homozygous zebrafish for all 19 single and 11 multi-gene knockouts were adult viable, indicating FA genes in zebrafish are generally not essential for early development. None of the mutant fish displayed gross developmental abnormalities except for fancp -/- fish, which were significantly smaller in length than their wildtype clutch mates. Complete female-to-male sex reversal was observed in knockouts for 12/17 FA genes, while partial sex reversal was seen for the other five gene knockouts. All adult females were fertile, and among the adult males, all were fertile except for the fancd1 mutants and one of the fancj mutants. We report here generation and characterization of zebrafish knockout mutants for 17 FA disease-causing genes, providing an integral resource for understanding the pathophysiology associated with the disrupted FA pathway.
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                Author and article information

                Contributors
                Journal
                Genes Dis
                Genes Dis
                Genes & Diseases
                Chongqing Medical University
                2352-4820
                2352-3042
                13 May 2022
                March 2023
                13 May 2022
                : 10
                : 2
                : 381-384
                Affiliations
                [a ]Center for Medical Genetics, Department of Biomolecular Medicine, Ghent University Hospital and Ghent University, 9000 Ghent, Belgium
                [b ]Department of Human Structure and Repair, Ghent University, 9000 Ghent, Belgium
                [c ]Cancer Research Institute Ghent (CRIG), Ghent University and Ghent University Hospital, 9000 Ghent, Belgium
                [d ]Department of Pathology, Ghent University and Ghent University Hospital, 9000 Ghent, Belgium
                Author notes
                []Corresponding author. Center for Medical Genetics, Department of Biomolecular Medicine, Ghent University and Ghent University Hospital, 9000 Ghent, Belgium. Kathleen.Claes@ 123456UGent.be
                Article
                S2352-3042(22)00121-0
                10.1016/j.gendis.2022.04.023
                10201593
                16556045-e175-4862-8bfa-face2a7cf6e7
                © 2022 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co., Ltd.

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 30 November 2021
                : 8 April 2022
                : 26 April 2022
                Categories
                Rapid Communication

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