Loss of the homologous recombination gene rad51 leads to Fanconi anemia-like symptoms in zebrafish

Abstract

RAD51 is an indispensable homologous recombination protein, necessary for strand invasion and crossing over. It has recently been designated as a Fanconi anemia (FA) gene, following the discovery of two patients carrying dominant-negative mutations. FA is a hereditary DNA-repair disorder characterized by various congenital abnormalities, progressive bone marrow failure, and cancer predisposition. In this report, we describe a viable vertebrate model of loss. Zebrafish loss-of-function mutants developed key features of FA, including hypocellular kidney marrow, sensitivity to cross-linking agents, and decreased size. We show that some of these symptoms stem from both decreased proliferation and increased apoptosis of embryonic hematopoietic stem and progenitor cells. Comutation of was able to rescue the hematopoietic defects seen in the single mutants, but led to tumor development. We further demonstrate that prolonged inflammatory stress can exacerbate the hematological impairment, leading to an additional decrease in kidney marrow cell numbers. These findings strengthen the assignment of as a Fanconi gene and provide more evidence for the notion that aberrant p53 signaling during embryogenesis leads to the hematological defects seen later in life in FA. Further research on this zebrafish FA model will lead to a deeper understanding of the molecular basis of bone marrow failure in FA and the cellular role of RAD51.

Multiplexed CRISPR/Cas9-mediated knockout of 19 Fanconi anemia pathway genes in zebrafish revealed their roles in growth, sexual development and fertility

“…To examine if loss of tp53 can rescue the sex reversal phenotypes as previously demonstrated for fancd1 , fancl and fancr [33–36], we introduced tp53 knockout mutation into fancp mutant fish as a representative of FA gene mutants with complete female to male sex reversal. First, tp53 mutant (hg91: c.368_374delCCGTGGT; p.S123Ffs*38) fish were generated using CRISPR-Cas9 method to target exon 5.…”

“…Three new FA genes ( FANCU , FANCV , FANCW ) were identified after initiation of our study. A knockout zebrafish model for the only autosomal dominant FA gene ( FANCR/RAD51 ) was also reported recently [36]. In addition to the 17 FA genes, our study targeted zebrafish homologs for two genes encoding FA-associated proteins ( FAAP24 and FAAP100 ), which are components of the FA core complex that facilitates the ID2 ubiquitination step of the DNA repair pathway.…”

“…A fancl mutant [33] was identified from an insertion-mutagenesis in a Tol2 transposon-mediated enhancer trap screen. Recently, a knockout for the autosomal dominant FA gene, fancr/rad51 , was generated using the ENU-mediated method [36]. The emergence of highly efficient CRISPR/Cas9 technology made it possible for us to efficiently test 19 genes.…”

“…We found that female-to-male sex reversal was a common phenotype in FA gene knockouts (Fig 5), which suggests that FA pathway plays an important role in zebrafish gonadogenesis. Interestingly, complete female-to-male sex reversal was reported in fancd1 , fancl and fancr knockout fish, and concomitant knockout of tp53 rescued the sex reversal phenotype in all three FA gene knockouts [33–36]. In fact, the sex reversal was demonstrated to be due to increased Tp53-mediated germ cell apoptosis at the critical time during sexual fate determination [33].…”

“…In FA patients, problems associated with gonadogenesis such as hypogonadism, and infertility are common, particularly male infertility [3, 64], and a recent study identified biallelic loss-of-function FANCM mutations as cause of non-obstructive azoospermia [3, 64]. Previous reports in zebrafish have shown that the homozygous knockouts of fancd1 [34, 35] and fancr [36] develop as infertile males with meiotic arrest in spermatocytes. It is surprising that infertility is not a common phenotype in many other FA gene knockout fish.…”