Global Gene Expression Atlas of Craniofacial Development (Research Project)

描述(申请人提供)：目标是创建一个全球基因表达图谱的头面部发育。中心论点是激光捕获显微切割和流式细胞仪与微阵列相结合可以有效地实现这一目标。具有基本完整的基因表示的微阵列可以用来快速确定激光捕捉颅面发育的显微解剖元素中每个基因的表达水平。因此，一个单独的实验就可以全面分析一种成分的基因表达状态，而检查每种结构和细胞类型的有限数量的实验就可以创建一份图谱。结合结构、凝集素染色和转基因GFP表达，将被用来精确识别特定的间隔和谱系，包括驱动神经脊诱导的细胞、鼻侧和凹陷、面部外侧和内侧隆起、神经脊和轴旁中胚层细胞、上颌和下颌隐窝、信号中心以及腭裂发生的结构。具体目标2，范围更有限，是制造SP8-GFP-Cre和Fgf8-GFP-Cre转基因小鼠工具，这将起双重作用，允许识别特定目标1和3的额外离散头面部成分，并帮助颅面研究界未来的区域特异性基因敲除研究。具体目标3是描述SP8突变小鼠的特征，该突变小鼠显示出颅面发育的巨大中断。我们推测SP8是成纤维细胞生长因子信号的上游，首先在前脑的内侧神经上皮细胞中，对驱动神经脊的增殖和迁移很重要，后来在鼻部的安慰剂和凹陷中，对面部隆起的形成很重要。我们提出了一种联合分子标记分析、基于Cre的SP8功能的遗传剖析以及基于芯片的野生型和SP8突变体信号中心的基因表达谱比较。 与公共卫生相关：唇腭裂是非常常见的出生缺陷，还有许多其他面部畸形，更严重，但不太常见。这些问题既有遗传原因，也有环境原因。我们建议使用最新的技术来研究涉及到脸部表情的所有基因，以更好地了解这一复杂的过程。人们希望这项工作将带来预防和/或治疗这些出生缺陷的新方法。

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.