Global Gene Expression Atlas of Craniofacial Development (Research Project)

描述（由申请人提供）：目的是创建颅面发育的全球基因表达图谱。中心论点是，激光捕获显微切割和流式细胞仪的组合，结合微阵列可以用来有效地实现这一目标。具有基本上完整的基因表达的微阵列可用于快速确定激光捕获颅面发育微解剖元件中每个基因的表达水平。因此，一个单一的实验，提供了一个组成部分的基因表达状态的综合分析和有限数量的实验检查每个结构和细胞类型可以创建一个图谱。结构、凝集素染色和转基因GFP表达的组合将用于精确鉴定特定的隔室和谱系，包括驱动神经嵴诱导的细胞、鼻基板和鼻凹、侧面和内侧面部隆起、神经嵴和近轴中胚层细胞、上颌骨和下颌骨凹陷、信号中心和腭发生的结构。具体目标2，范围更有限，是使Sp 8-GFP-Cre和Fgf 8-GFP-Cre转基因小鼠工具，这将服务于双重目的，以允许识别额外的离散颅面组件的具体目标1和3，并帮助颅面研究界未来的域特异性基因敲除研究。具体目标3是表征Sp 8突变小鼠，其显示颅面发育的大规模破坏。我们假设，SP 8是上游的FGF信号首先在内侧神经上皮细胞的前脑，重要的驱动神经嵴增殖和迁移，后来在鼻基板和凹，重要的信号形成面部隆起。我们提出了一个组合的分子标记分析，一个Cre为基础的Sp 8功能的遗传解剖，和一个基于微阵列的基因表达谱比较野生型和Sp 8突变体中的信号中心。

DESCRIPTION (provided by applicant): The objective is to create a global gene expression atlas of craniofacial development. The central thesis is that a combination of laser capture microdissection, and FACS, combined with microarrays can be used to efficiently achieve this goal. Microarrays with essentially complete gene representation can be used to rapidly determine the expression levels of every gene in laser capture microdissected elements of craniofacial development. A single experiment, therefore, provides a comprehensive analysis of the gene expression status of one component and a limited number of experiments examining each structure and cell type can create an atlas. A combination of structure, lectin staining, and transgenic GFP expression will be used to precisely identify specific compartments and lineages, including cells driving neural crest induction, nasal placodes and pits, lateral and medial facial eminences, neural crest and paraxial mesoderm cells, maxilla and mandibular recesses, signaling centers, and the structures of palatogenesis. Specific Aim 2, more limited in scope, is to make Sp8-GFP-Cre and Fgf8-GFP-Cre transgenic mouse tools, which will serve a dual purpose, to allow identification of additional discrete craniofacial components for Specific Aims 1 and 3, and to aid future domain specific gene knockout studies by the craniofacial research community. Specific Aim 3 is to characterize the Sp8 mutant mouse, which shows a massive disruption of craniofacial development. We hypothesize that Sp8 is upstream of FGF signaling first in the medial neuroepithelium of the forebrain, important for driving neural crest proliferation and migration, and later in the nasal placodes and pits, important for signaling the forming facial eminences. We propose a combined molecular marker analysis; a Cre based genetic dissection of Sp8 function, and a microarray based gene expression profile comparison of signaling centers in wild type and Sp8 mutants.