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解剖和FACS结合微阵列可以有效地实现这一目标。具有基本完整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素中每个基因的表达水平。一个单一的实验，因此，提供了一个成分的基因表达状态的全面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表达的结合将用于精确识别特定的区室和谱系，包括驱动神经嵴诱导的细胞、鼻座和鼻窝、外侧和内侧面部隆起、神经嵴和旁轴中胚层细胞、上颌和下颌凹陷、信号中心和腭形成结构。特异性目标2，范围更有限，是制作Sp8-GFP-Cre和Fgf8-GFP-Cre转基因小鼠工具，这将有双重目的，允许鉴定额外的离散颅面成分，用于特异性目标1和3，并帮助未来颅面研究界进行区域特异性基因敲除研究。特异性目的3是表征Sp8突变小鼠，其表现出颅面发育的大规模中断。我们假设Sp8是FGF信号的上游，首先在前脑的内侧神经上皮，对驱动神经嵴的增殖和迁移很重要，然后在鼻基质和凹处，对面部隆起的形成很重要。我们建议联合分子标记分析；基于Cre的Sp8功能基因解剖，以及基于微阵列的Sp8野生型和Sp8突变体信号中心基因表达谱比较。

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. PUBLIC HEALTH RELEVANCE: Cleft lip and palate are very common birth defects, and there are many other facial malformations that are more severe, but less common. These problems have both genetic and environmental causes. We propose to use the latest technologies to study all of the genes involved in making the face, to gain a better understanding of this complicated process. It is hoped this work will lead to new methods to prevent and/or treat these birth defects.