Testing the Role of Wnt8a Diffusion in Wnt Mediated Neural Posteriorization

This study assesses the role of diffusion of Wnt8a in patterning the anterior posterior (AP) axis of the neural plate. The vertebrate neural plate is subdivided into different morphological domains that define different functional regions. Failure to establish these morphological domains in the correct size have been shown to be associated with neuropathologies such as schizophrenia. While the adult brain is highly complex morphologically it begins as a simple sheet of cells. The neural ectoderm is first established on the dorsal side of the embryo, being activated from non-neural ectoderm by the presence of low levels of Bmp signaling. The neural plate initially lacks any AP pattern and will take on a forebrain fate if no additional signals are received. One of the key genes in establishing early posterior neural fate is wnt8a. wnt8a is expressed in the margin of the embryo and has been shown to posteriorize the neural plate in a dose dependent manner, with higher levels of Wnt signaling inducing a more posterior fate. Wnts are lipoproteins that are secreted. However, their hydrophobic nature due to their lipidation has raised the question of how Wnt8a is able to diffuse across the neural plate to establish a posteriorizing gradient. Recent experiments in Drosophila Melanogaster have suggested that membrane tethered variants of wg, the Wnt homolog, can still pattern the wing disc. Our research was focused on testing if diffusion was necessary in vertebrate neural patterning. We generated two transgenic lines, Tg(otx2FM:h2bcitrine) and Tg(en2aDRE:egfp) to allow imaging of the developing neural plate during epiboly. Neither of these transgenes were expressed at sufficient levels during early neural patterning, making them inappropriate for this study. However, the expression pattern of Tg(en2aDRE:egfp) suggests a novel positive feedback loop in the regulation of en2a. Testing of the Wnt8a diffusion hypothesis using a membrane tethered variant of wnt8a did not posteriorize the neural plate, but instead affected cell/cell cohesion and trunk development, an effect that was not predicted. While our research did not answer the question of the necessity of Wnt8a diffusion we did expand our knowledge of the regulation of neural patterning genes