Abstract
Mild mutations in BRCA2 (FANCD1) cause Fanconi anemia (FA) when homozygous, while severe mutations cause common cancers including breast, ovarian, and prostate cancers when heterozygous. Here we report a zebrafish brca2 insertional mutant that shares phenotypes with human patients and identifies a novel brca2 function in oogenesis. Experiments showed that mutant embryos and mutant cells in culture experienced genome instability, as do cells in FA patients. In wild-type zebrafish, meiotic cells expressed brca2; and, unexpectedly, transcripts in oocytes localized asymmetrically to the animal pole. In juvenile brca2 mutants, oocytes failed to progress through meiosis, leading to female-to-male sex reversal. Adult mutants became sterile males due to the meiotic arrest of spermatocytes, which then died by apoptosis, followed by neoplastic proliferation of gonad somatic cells that was similar to neoplasia observed in ageing dead end (dnd)-knockdown males, which lack germ cells. The construction of animals doubly mutant for brca2 and the apoptotic gene tp53 (p53) rescued brca2-dependent sex reversal. Double mutants developed oocytes and became sterile females that produced only aberrant embryos and showed elevated risk for invasive ovarian tumors. Oocytes in double-mutant females showed normal localization of brca2 and pou5f1 transcripts to the animal pole and vasa transcripts to the vegetal pole, but had a polarized rather than symmetrical nucleus with the distribution of nucleoli and chromosomes to opposite nuclear poles; this result revealed a novel role for Brca2 in establishing or maintaining oocyte nuclear architecture. Mutating tp53 did not rescue the infertility phenotype in brca2 mutant males, suggesting that brca2 plays an essential role in zebrafish spermatogenesis. Overall, this work verified zebrafish as a model for the role of Brca2 in human disease and uncovered a novel function of Brca2 in vertebrate oocyte nuclear architecture.
Highlights
People who are heterozygous for strong mutations in the tumor suppressor gene BRCA2(FANCD1) have increased susceptibility to breast, ovarian, prostate, and pancreatic cancers [1,2,3]
People with two mild BRCA2(FANCD1) mutations develop Fanconi Anemia, which reduces DNA repair leading to genome instability, small gonads, infertility, and cancer
We discovered that zebrafish brca2 mutants show chromosome instability and small gonads, and they develop only as sterile adult males
Summary
People who are heterozygous for strong mutations in the tumor suppressor gene BRCA2(FANCD1) have increased susceptibility to breast, ovarian, prostate, and pancreatic cancers [1,2,3]. Breast cancer risk for females heterozygous for germline mutations in BRCA2 is nearly 60% by age 50 [4] and for ovarian cancer is 11% [5]. Biallelic inheritance of hypomorphic BRCA2 mutations in the germline results in Fanconi anemia (FA), a disease characterized by catastrophic anemia, genome instability, characteristic morphological defects, and enormously elevated risk for leukemia (800 fold) and squamous cell carcinomas (2000 fold) [12,13,14]. The BRCA2 subtype of Fanconi anemia represents complementation group D1 [15] and results in a severe form of the disease with nearly 100% incidence of leukemia and/or solid tumors by 5 years of age [16,17]. HDR helps repair DNA breaks associated with meiosis, and mouse mutants in FA genes have defects in meiotic cells [19]. Zebrafish fancl mutants experience female-to-male sex reversal due to the apoptotic loss of meiotic oocytes at the time of sex determination [20], consistent with the abnormal activation of the apoptotic pathway in the absence of Fanconi gene activity [21]
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