Idenfication of miRNAs involved midfacial development and clefting

描述（由申请人提供）：本项目的目标是鉴定参与脊椎动物中面部发育的microRNA（miRNAs），并确定它们在这一过程中的功能。我们的假设是，有限数量的miRNAs通过特定蛋白编码基因的精确时空表达，在面中部的模式化、增殖和分化中发挥着至关重要的作用。此外，我们假设这些miRNAs的功能丧失和获得导致面中部和口面缺陷/裂缝。在人类中，面部中部发育缺陷，包括唇腭裂，占每年出生缺陷的最大数量。仅唇腭裂就有400多种综合征，其发生率为1/800活产。我们的实验室有一个长期的兴趣，在确定基因调控网络，控制正常的口面发育，其中许多是在动物模型中的面中部裂的原因。在我们的初步数据中，我们表明，微阵列技术可以产生全面的miRNA表达谱，生物信息学分析可以定义其表达模式在脊椎动物中是保守的miRNA。在本提案中，我们将在三个特定目标中研究发育中面部中表达的miRNA的身份和功能。目的1：利用miRNA微阵列技术，确定并验证小鼠上颌/额鼻发育过程中miRNA的时空表达模式。在目标2中，我们将确定在小鼠和斑马鱼胚胎中鉴定的miRNAs的原位表达模式，以定义那些显示保守表达模式的miRNAs。在目标3中，我们将使用斑马鱼通过功能丧失和获得分析来确定miRNA的调节功能和形态发生作用模式。我们建议同时使用小鼠和斑马鱼系统，因为它们互补的成熟的实验和遗传方法可以对这一过程进行详细的分析。通过定义参与小鼠中面部发育的miRNA，然后快速有效地确定斑马鱼中miRNA的功能，我们将能够阐明参与脊椎动物中面部发育的miRNA。这对于我们进一步了解导致人类中面部出生缺陷综合征的遗传事件至关重要。 公共卫生相关性：面中部出生缺陷是世界范围内一个重大的社会和医学问题。通过定义参与面中部发育的miRNAs，我们期望揭示参与正常和异常面中部发育的新的遗传调控机制。我们对面部中部miRNAs的表征将为开发新的治疗方法提供潜在的靶点，目的是修复和预防面部中部人类先天性缺陷。

DESCRIPTION (provided by applicant): The goal of this project is to identify microRNAs (miRNAs) involved in vertebrate mid-face development and to determine their function in this process. Our hypothesis is that a limited number of miRNAs play crucial roles in the patterning, proliferation and differentiation of the mid-face through precise spatial and temporal expression of specific protein-coding genes. Further, we hypothesize that both loss- and gain-of-function of these miRNAs leads to mid-facial and orofacial defects/clefting. In humans, defects in mid-facial development, including cleft lip/palate, account for the largest number of birth defects annually. Alone, over 400 syndromes result in cleft lip and palate, which has an occurrence rate of 1 in 800 live births. Our laboratories have a long-standing interest in determining the gene regulatory networks that control normal orofacial development, many of which are causative in animal models of mid-facial clefting. In our Preliminary Data, we show that microarray technology can generate comprehensive miRNA expression profiles and that bioinformatic analysis can define miRNAs whose expression patterns are conserved across vertebrates. In this proposal, we will investigate the identity and function of miRNAs expressed in the developing mid-face in three Specific Aims. In Aim 1, we will determine and validate the temporal and spatial expression patterns of miRNAs in the developing mouse maxillary/frontonasal prominences using miRNA microarray technology. In Aim 2, we will determine the in situ expression patterns of identified miRNAs in both mouse and zebrafish embryos to define those that show a conserved pattern of expression. In Aim 3, we will use zebrafish to determine the regulatory function and morphogenetic mode of action of the miRNAs by loss- and gain-of-function analysis. We propose using both mouse and zebrafish systems because their complementary well-established experimental and genetic methods make a detailed analysis of this process feasible. By defining miRNAs involved in mouse mid-facial development and then quickly and efficiently determining miRNA function in zebrafish, we will be able to elucidate the miRNAs involved in vertebrate mid-facial development. This is crucial to further our understanding of genetic events leading to human mid-facial birth defect syndromes. PUBLIC HEALTH RELEVANCE: Mid-facial birth defects represent a significant social and medical problem throughout the world. By defining the miRNAs involved in mid-facial development, we expect to uncover novel genetic regulatory mechanisms involved in normal and abnormal mid-face development. Our characterization of miRNAs in the mid-face will provide potential targets for the development of novel therapeutics, with the goal of repairing and preventing mid-facial human congenital defects.