Fig. 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. atmcmg33/cmg34;tp53zdf1/zdf1 mutant zebrafish are prone to develop tumours. (F) Age of tumour onset is significantly lower in atmcmg33/cmg34;tp53zdf1/zdf1 zebrafish compared to atm+/-;tp53zdf1/zdf1 and atm+/+;tp53zdf1/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.
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