Molecular Characterization of Zebrafish Colgate, A Potent WNT Antagonist

Ohio State University Research Foundation -Do Not Use, Columbus OH

Investigators

Abstract

0315765 Henion The determination of the vertebrate body plan occurs during gastrulation as the germ layers are induced and patterned along the dorsoventral (DV) and anteroposterior (AP) axes. The process of dorsal specification is initiated soon after fertilization and relies on the functioning of an inductive signaling center called the gastrula organizer. The organizer forms as the result of nuclear localization of b-catenin and the inductive signaling of downstream genes. Once the organizer is formed, it can dorsalize lateral mesoderm and induce and pattern neural fates in the ectoderm. These activities can, at least in part, be attributed to its ability to inhibit the effects of the BMP, Nodal and Wnt families of secreted factors. However, the molecular control of these complex induction and patterning events is incompletely understood. The zebrafish mutant colgate (col) is a recessive, embryonic lethal mutation with visible defects in mesodermal and neural structures. Further analysis revealed a reduction in dorsal mesodermal derivatives such as the heart and an expansion of ventral mesodermal derivatives including blood. The central nervous system is smaller overall, and is regionally posteriorized. Examination of col mutant embryos during early gastrulation stages revealed an expansion of wnt8-expressing lateral mesoderm and a reduction in the expression domains of organizer-specific genes. In addition, wnt8b expression is expanded later in gastrulation in col mutant embryos. Inhibition of Wnt8 signaling by antisense morpholino-mediated gene knockdown in col mutant embryos rescues the organizer, DV patterning and anterior neuroectoderm phenotypes, but does not rescue the regionalized AP patterning defects in the brain. In contrast, Wnt8b morpholino injection does not rescue the early defects in col mutant embryos, but does rescue the regionalized AP brain patterning defects. Taken together, these results suggest that the gene encoded by the col locus is normally required for maintenance of the organizer and correct DV and AP patterning via antagonism of Wnt8 and Wnt8b signaling. This proposal includes experiments designed to determine the cell autonomy of the col mutation, identify the col gene by positional cloning, determine the expression pattern of col during embryogenesis and determine the effects of col misexpression on development. Taken together, these results will identify the col gene and provide important insights into its normal functions during development in the specification of the vertebrate body plan and brain patterning. This knowledge in turn will form the basis for future genetic and molecular characterization of the function of col in the regulation of Wnt-signaling. Lastly, given the prominent role of members of the Wnt-signaling pathway in tumorigenesis and tumor suppression and the emerging capability of comparative genomics, these results are likely to provide insights into clinically relevant conditions in humans. This proposal directly and indirectly provides for substantial training and experience opportunities for students at various stages of education, including graduate students, undergraduates and local high school students. In addition to the obvious importance of support for graduate students, support and opportunities for undergraduates and high school students is particularly significant for providing positive experiences in research science which they may either subsequently decide to pursue or at least share with the broader lay community.