Chemical Genetic Studies of Tumor Suppressors in the Developing Zebrafish

The zebrafish is a powerful disease model system. Its distinct advantages allow it to be used for high-throughput genetic and chemical screens with high translational potential. One mutant isolated from a screen for cell cycle deficiencies is the zebrafish cdc25a mutant standstill. This mutant suffers from a G2/M arrest due to activation of the checkpoint protein ATM such that ATM knockdown or inhibition rescues the cell cycle. Additional knockdown studies reveal that the unique zebrafish cdc25 gene cdc25d is responsible for cell cycle progression in the absence of functional Cdc25a or ATM. Assays to measure genomic stress reveal that ATM is activated in the absence of DNA damage. Activation of ATM in the absence of DNA damage is a unique occurrence, but further studies seeking regulators of ATM reveal that ATM is constitutively active at a basal level. This basal ATM activity is sufficient to attenuate cell cycle rate. Other studies involving the use of zebrafish to study cancer involve a screen using peptoids. The peptoid is a class of compound that is modular, easy and inexpensive to synthesize, and stable in vivo. Initial tests reveal that peptoids are able to enter the zebrafish embryo, remain stable, and exert biological functions. Screening of 3,744 compounds for peptoids that specifically affect zebrafish mutant for p53 reveal a group of 4 structurally similar peptoids. Further studies of these peptoids reveal that they are able to perform their function on mouse embryonic fibroblasts in vitro. Finally, I sought the role of the DNA damage marker H2AX in early zebrafish development. While studying the DNA damage response in cdc25a mutants, I discovered that H2AX is present and cyclic in pre-midblastula transition embryos. This phosphorylation is necessary for synchronization and relies on the ability of ATM to phosphorylate H2AX on Ser-139 such that inhibition of ATM or ablation of the phosphorylation site leads to desynchronization and improper development.