Abstract
Investigating the in vivo role of tumor suppressor genes in cancer is technically challenging due to their essential requirement during early animal development. To address this bottleneck, we generated genetic mosaic adult zebrafish using TALEN genome editing and demonstrate somatic inactivation of the tumor suppressor retinoblastoma1 (rb1) induces tumorigenesis at high frequency. 11–33% of 1-cell stage embryos injected with TALEN mRNAs targeting rb1 exon 2 or 3 develop tumors beginning as early as 3.5 months of age. Lesions predominantly arise in the brain and show features of neuroectodermal-like and glial-like tumors. Mutant allele analysis is consistent with tumor initiation due to somatic inactivation of rb1, revealing a conserved role for rb1 in tumor suppression across vertebrates. In contrast to genetic mosaics, heterozygous rb1−/+ adults show no evidence of neoplasia, while homozygous mutant rb1−/− are larval lethal. This is the first demonstration that somatic inactivation of a tumor suppressor causes cancer in zebrafish, and highlights the utility of site-specific nucleases to create genetic mosaic zebrafish for tumor suppressor gene discovery. Somatic inactivation with site-directed nucleases in zebrafish presents a rapid and scalable strategy to study tumor suppressor gene function in cancer.
Highlights
Senescence, apoptosis, DNA repair, and chromosome integrity[13,14]
We present a proof of principle study in zebrafish that shows genome editing nucleases can be used to study tumor suppressor gene function by targeted somatic inactivation
We demonstrate that TAL effector nucleases (TALENs)-mediated targeting of rb[1] results in highly penetrant tumor formation in genetic mosaic adults
Summary
We used TALEN targeting to isolate germline mutations in the zebrafish retinoblastoma[1] (rb1) tumor suppressor gene. During the course of this study, we observed that the genetic mosaic adults generated by somatic inactivation of rb[1] in embryos develop tumors in the head region at high frequency (Fig. 1). Serial sections of 14/52 dissected brains in eight exon 2-targeted and six exon 3-targeted fish revealed 12 neoplastic mass lesions that infiltrated and effaced the neuropil and two brains with expanded glial rests (Table 1). Two brain tumors from exon 2 and exon 3 targeted fish were analyzed by immunohistochemistry for identification of proliferating cells and expression of glial and neuronal markers. The overall structure appeared mostly normal, but the lateral division of the valvula cerebelli contained numerous proliferating cells not observed in wild type (Supplementary Figure 2). Images of glial and neuronal differentiation markers labeling wild type brain and a representative brain
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
