Idenfication of miRNAs involved midfacial development and clefting

描述(申请人提供)：本项目的目标是确定参与脊椎动物面部中部发育的microRNAs(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通过特定蛋白质编码基因的精确空间和时间表达，在面部中部的图案化、增殖和分化中发挥关键作用。此外，我们假设这些miRNAs功能的丧失和获得都会导致面部中部和口面部的缺陷/裂隙。在人类中，包括唇腭裂在内的面部中部发育缺陷是每年出生缺陷数量最多的原因。仅此一项，就有400多种综合征导致唇腭裂，其发生率为每800名活产儿中就有1名。我们的实验室长期以来一直对确定控制正常口腔面部发育的基因调控网络感兴趣，其中许多网络在面部中部裂伤的动物模型中是致病的。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在脊椎动物中保守的表达模式。在这个提案中，我们将从三个特定的目的来研究在发育中面部表达的miRNAs的身份和功能。在目标1中，我们将利用miRNA微阵列技术来确定和验证miRNAs在发育中的小鼠上颌骨/额鼻突中的时空表达模式。在目标2中，我们将确定已识别的miRNAs在小鼠和斑马鱼胚胎中的原位表达模式，以确定那些显示保守表达模式的miRNAs。在目标3中，我们将利用斑马鱼通过功能丧失和功能获得的分析来确定miRNAs的调控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，因为它们相互补充、完善的实验和遗传方法使详细分析这一过程是可行的。通过定义参与小鼠面中部发育的miRNAs，然后快速有效地确定斑马鱼的miRNA功能，我们将能够阐明参与脊椎动物面中部发育的miRNAs。这对于进一步了解导致人类面部中部出生缺陷综合征的基因事件至关重要。 与公共卫生相关：面部中部出生缺陷是全世界一个重大的社会和医学问题。通过定义参与面部中部发育的miRNAs，我们有望揭示涉及正常和异常面部中部发育的新的基因调控机制。我们对面中部miRNAs的鉴定将为开发新的治疗方法提供潜在的靶点，目的是修复和预防人类面部中部的先天性缺陷。

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.