Loss of the homologous recombination generad51leads 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 RAD51 loss. Zebrafish rad51 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 p53 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 RAD51 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.

High-throughput generation and phenotypic characterization of zebrafish CRISPR mutants of DNA repair genes

“…To fully understand functional annotations of genes in vivo, we screened the phenotypes of the mutants in embryogenesis, viability, growth, sex determination, hematopoiesis and sensitivity to DNA damaging agents. All the 33 homozygous mutants grew normally until 48 hpf with only four mutants showing morphological malformation and hematopoietic defects before 6 dpf, in line with observations that genomic homeostasis is linked to hematopoiesis (Farres et al 2015;Botthof et al 2017;Marsh et al 2018). Mutants of pcna and atad5a, two critical genes for DNA replication (Kang et al 2019a), showed reduced proliferation and induced apoptosis in the blood precursors at the CHT, while ddb1 and pcid2 homozygous mutants showed loss of HSPCs caused by reduced proliferation.…”

“…In addition, our six zebrafish mutants showed female-to-male sex reversal defects; blm -/-, brca2 -/-, fanci -/-, rad51 -/-, rad54l -/and rtel1 -/-. While rad54l -/and rtel1 -/mutants demonstrated a possible sex reversal phenotype with normal fertility like fanci -/homozygotes (Ramanagoudr-Bhojappa et al 2018), blm -/male animals were infertile without normal functional germ cells in the testis like brca2 -/and rad51 -/where primordial germ cells failed to survive and proliferate in the testes (Shive et al 2010;Botthof et al 2017). Although Blm homozygous KO mouse embryos were developmentally delayed and died by embryonic day 13.5 (Chester et al 1998), blm cu55/cu55 zebrafish survived and we were able to observe aberrant spermatogenesis, potentially recapitulating a phenotype of human male infertility in patients with Bloom syndrome (de Renty and Ellis 2017).…”

“…Previous studies demonstrated that zebrafish mutants of HR and FA genes failed to generate female animals due to sex reversal defects without normal germ cell development (Shive et al 2010;Botthof et al 2017;Ramanagoudr-Bhojappa et al 2018). To examine whether the DNA repair genes investigated here are involved in sex determination and germ cell development, we screened sexes of the 21 homozygous mutants that survive to 4-6 month of age (Supplemental Fig.…”

“…Although several studies have suggested that many DNA repair and replication genes play a key role in hematopoiesis and human blood disorders (Xu et al 2006;Alvarez et al 2015;Gao et al 2015;Botthof et al 2017;Marsh et al 2018), their functions in hematopoietic progenitor development is not well understood. To understand how HSPC development is affected in our DNA repair mutant library, we examined expression of HSPC marker cmyb by whole-mount in situ hybridization (WISH).…”

Scaling up to study brca2: the zeppelin zebrafish mutant reveals a role for brca2 in embryonic development of kidney mesoderm

“…Deficiency of another critical member of this complex, rad51 , has recently been studied in zebrafish and shown to display symptoms of FA and smaller kidneys [78] . In light of this, other gene members of the FA pathway should be more closely scrutinized in the context of podocyte and kidney development to assess whether diminished DNA repair by other FA genes is causative of the fate switch seen between podocytes and IR in zep mutants.…”