A molecular and systems biology analysis of pleiotropic vertebrate phenotypes.

摘要

Mutations can contribute to the tissue overgrowth, disorganization and cellular atypia characteristic of cancer. This has guided current research in the direction of discovering new mutations in genes that govern the transformation of normal human cells into malignant ones. A mutagenesis screen in zebrafish recovered the mutant huli hutu (hht), whose pleiotropic, histological phenotype included the kind of tissue disorganization and nuclear atypia that can be seen with precancers and cancer. Identification of such mutations may contribute to our understanding of cytologic cancer phenotypes, and possibly produce additional models for aspects of human disease, including diseases of the eye and gastrointestinal tract.;High resolution recombinant mapping using simple sequence repeat markers, generated five candidate genes: dimt1l, pola2, mier3, mrsp36, and cenph. Morpholino antisense knockdown, transcript analysis, and direct sequencing of complementary and genomic DNA revealed a nonsynonymous substitution in the C-terminal domain of Pola2, the B subunit of DNA polymerase alpha-primase. Immunohistochemical staining with cell proliferation markers BrdU and pH3 and TUNEL analysis suggests defects in proliferation, resulting in tissue-specific apoptosis. Ultrastructural analysis reveals a heterochromatin distribution consistent with cells responding to DNA damage.;The multiple-organ phenotype seen with hht inspired a genome-wide analysis of pleiotropic phenotypes observed in a collection of zebrafish insertional mutants.;Knowledge of pleiotropic phenotypes caused by single gene deficiencies requires characterization extending beyond single organs. However, the proportion of vertebrate genes that function across tissue boundaries is unknown due to the paucity of sensitive and systematic studies in whole model organisms. To address this combinations of anatomical and histological phenotypes were evaluated in 97 zebrafish insertional mutants with known single gene deficiencies. Eleven mutants exhibited defects in a single organ. Eighty-three had a detectable phenotype in two or more organs, and 72 in three or more. The results suggest that a majority of developmental genes in vertebrates function in multiple tissues, and that phenotypic analyses that cross tissue boundaries are required for more comprehensive understanding of gene function. This type of analysis can be applied across other model systems to make functional annotation of metazoan genes more complete, and may be extended to drug discovery and assessment of environmental toxicity.