The Role of Wnt Lipid Modifications in Chick Neural Tube Proliferation and Patterning

Wnt proteins play important roles in regulating cell-cell interactions during embryonic development. Mutations in Wnt genes or the Wnt signaling pathway lead to developmental defects, human diseases, and cancer. In our lab, we seek to understand the mechanisms by which the dorsal to ventral gradient of Wnt1/3a activity regulates the expansion and patterning of the neural tube. Previous studies show that overexpression of Wnt1 increases proliferation and promotes specification of dorsal interneurons. It has also been shown that two conserved Wnt residues (C93 and S224 in Wnt1) are lipid modified. Though these residues are important for the secretion of Wnt1, only S224 is absolutely required for Wnt1 signaling via the beta-catenin dependent pathway in in vitro studies. In this in vivo experiment, we extend our previous results by electroporating mutated Wnt1 (C93A or S224A) into the chick neural tube using a plasmid called pCIG, and comparing the effect on proliferation and neuronal specification on the control and experimental side of the embryo. We hypothesize that because S224 is absolutely required for bet-catenin dependent signaling in HEK293T cells, electroporated Wnt1 S224A will have no effect on proliferation and specification of dorsal interneurons. By contrast, since C93A retains partial activity in HEK293T cells, we expect to see a small, but significant increase with respect to proliferation and dorsal interneuron specification. We plan to observe these changes using immunohistochemical staining techniques specific for mitotic cells and dorsal interneuron subpopulations. A confocal microscope will be used to visualize stained sections.