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Abstract
Apurinic/apyrimidinic endonuclease 1, a key enzyme in repairing abasic sites in DNA, is an embryonic lethal in mice. We are examining its role in embryogenesis in zebra fish. Zebra fish contain two genomic copies (zfAPEX1a and zfAPEX1b) with identical coding sequences. zfAPEX1b lacks introns. Recombinant protein (ZAP1) is highly homologous with and has the same enzymatic properties as its human orthologue. ZAP1 is highly expressed throughout development. Embryos microinjected with morpholino oligonucleotide (MO) targeting the translation start site die at approximately the midblastula transition (MBT) without apoptosis. They are rescued with mRNA for human wild-type APEX1 but not for APEX1 encoding endonuclease-defective protein. Rescued embryos develop dysmorphic hearts, pericardial edema, few erythrocytes, small eyes, and abnormal notochords. Although the hearts in rescued embryos form defective loops ranging from no loop to one that is abnormally shaped, cardiac myosin (cmlc2) is present and contraction occurs. Embryos microinjected with MO targeting zfAPEX1a intron-exon junctions also pass the MBT with similar abnormalities. We conclude that AP endonuclease 1 is involved in both repairing DNA and regulating specific early stages of embryonic development.
We thank P. Yelick for providing the zebra fish cDNA library in pSPORT; P. Yelick, H. Sive, and H. Jo for technical expertise and helpful discussions; and H. Sive and Z. Werb for critical reading of the manuscript. We thank C. Geoffrey Burns for providing cmlc2-GFP fish. Nomarski and fluorescence microscopy was performed with the W. M. Keck 3D fusion microscope at Northeastern University's Keck facility.
This study was supported by NIH CA74202, NIH HD053317, and funds from Northeastern University.