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 ectoderm shortly after it 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. One aim of this grant is to elucidate the relationship of Six1, 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 Six1 in establishing the lateral neural ectoderm, that gives rise to the placodes and how does it specifically relate to other NFS-genes? In the parent grant we proposed to study the relationship of NFS genes to one that we cloned, Six1 (Brugmann et al., 2004). Six1 expands the placodal ectoderm and holds it in an immature state. In the original grant, we proposed to study whether Six1 activates or suppresses the expression of 6 known lateral 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. Six1, a sine oculis transcription factor which is expressed in the early placodal ectoderm that gives rise to the cranial PNS, is a key regulator of neural placodal fate (Brugmann et al., 2004, Development). Being a newly cloned gene, we hypothesize that we will identify numerous downstream targets of Six1 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 Six1. Six1 mRNA (400pg) 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 Amphibian�s Medium. When ACs reach stage 10.5, an early step in neural ectoderm specification, they will be cultured in 200ng/nl Noggin protein (R&D Systems) to induce the expression of neural plate genes. When ACs reach stage 16, at which time the placodes have formed, 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, also treated with Noggin, derived from sibling embryos. We will repeat the experiment 5 times (total of 10 samples). Extracted RNA will be evaluated for quality, reverse transcribed into cDNA, amplified, fragmented and then biotinylated using the NuGEN Ovation Biotin System kit.
