Role of maternal mRNA degradation in whole-body cellular phenotypes caused by DNA polymerase α deficiency

A histology-based zebrafish forward genetic screen for mutations causing cytological abnormalities yielded a mutant, huli hutu (hht), whose pleiotropic phenotype includes atypical nuclei in gastrointestinal cells and nuclear fragmentation in the retina and central nervous system. We have used a new, 3D form of histology based on micro-CT, X-ray histotomography, to reveal diminished heart volume and shape, and dysplastic cartilage and skin. We were also able to definitively establish the absence of a swim bladder and pneumatic duct, which is challenging to determine usig histology. Cell death shown by karyorrhexis was associated with DNA damage identified by ɤ-H2AX stainng in the brain, eyes, and spinal cord. These phenotypes were caused by a frameshift-based premature stop codon at the 38th amino acid position of the 600-amino acid Pola2 protein, or B subunit of DNA polymerase α (Pol α). An extended 120-168 hpf survival of the hht fish stands in striking contrast to the immediate cell cycle arrest of the corresponding mutants in yeast and Arabidopsis. We found that the prolonged lifespan of these mutants can be explained by the presence of wild-type maternal pola2 mRNA in the fertilized embryos. The gradual disappearance of wild-type mRNA in homozygous mutant embryos appears to cause diminished DNA synthesis, increased DNA damage, cell death, and tissue-dependent cytological deformities. The breadth of cytological, tissue, and organ phenotypes of hht mutants can be used as a model for developing computational tools for quantitative 3D characterization of pleiotropy, including cytological and tissue phenotypes across organ systems.