ABSTRACT: The goal of the current NS23158 funding is to understand how neural fate-stabilizing (NFS) genes function in order to provide fundamental knowledge about their potential roles in neural stem cell development. Neural fate-stabilization is characterized by the expansion of the neural plate shortly after the presumptive neural ectoderm has been induced by the repression BMP signaling. During this time period a number of early-expressed NFS genes are expressed, each of which expands the neural plate in gain-of-function assays. But, we do not know how these genes are related to one another or whether they are arranged in a linear gene pathway or multi-path hierarchy. A key aim of this grant is to elucidate the relationship of foxD5, an early-expressed NFS gene that we cloned, to other NFS-genes. We propose to greatly enhance this analysis by utilizing DNA microarray analyses to identify unsuspected and novel target genes. What is the function of foxD5 in neural fate-stabilization, and how does it specifically relate to other genes in the early-expressed group? In the parent grant we proposed to study the relationship of early-expressed NFS genes to one that we cloned, FoxD5 (Sullivan et al 2001). FoxD5 expands the neural plate and holds it in an immature state. In the original grant, we proposed to study whether foxD5 activates or suppresses the expression of 6 known early-expressed NFS genes, using PCR and in situ hybridization of animal caps and whole embryos. Herein we propose to use DNA microarray technology to reveal a much broader spectrum of potential target genes. FoxD5, a fork-head transcription factor which is expressed in the early neuroectoderm, is a key regulator of neural plate fate during expansion of the neural plate. Being a newly cloned gene, we hypothesize that we will identify numerous downstream targets of FoxD5, by the proposed microarray analyses. This information will allow us to study their function by gain- and loss-of function studies in the whole embryo. Animal cap (AC) explants are a naïve embryonic ectoderm that is removed from embryonic signaling centers. They allow one to perform gene induction assays in the absence of confounding growth factors, and thus are ideal for identifying downstream targets of transcription factors. In this experiment we will take a gain-of-function approach to identify which genes are induced/repressed by foxD5. FoxD5 mRNA (200pg) will be injected at the 2-cell stage, and embryos cultured until stage 8, at which time the zygotic genome begins transcription. AC explants will be cut from the embryos and cultured in Normal Amphibians Medium. When ACs reach stage 10.5, an early step in neural ectoderm specification, they will be snap frozen in liquid nitrogen and RNA purified according to the Qiagen Rneasy Protect kit protocols. Our preliminary experiments indicate that 8-10ug of total RNA can be recovered with high purity from ~150 ACs with this method. As controls, RNA will be purified from uninjected animal caps derived from sibling embryos. If foxD5 represses NFS gene expression, it will not be detected in the above experiment because NFS genes are not expressed in AC explants in the absence of neural induction. Therefore, we will repeat the experiment but additionally treat the ACs (FoxD5-injected and sibling uninjected) at stage 8 with Noggin protein (R&D Systems) to induce neural ectoderm. For all four sample sets, RNA will be reverse transcribed, amplified and biotinylated using the NuGEN Ovation kit. Keywords: neural fate, FoxD5, Noggin
